1,2-dioxetane derivatives and reagents employing them

ABSTRACT

A 1,2-dioxetane derivative of the formula (I): 
                         
wherein Ar is an aryl group which may have an alkyl group, an aryl group, a halogen atom, an alkoxyl group, a carboxyl group, a formyl group, an alkyl ester, an aryl ester, an alkylketone, an arylketone or a hetero ring bonded thereto, X is a substituent capable of labeling an organic compound or a biological molecule, or an ester, Y is a hydrogen atom, an acyl group or a group of the formula —Si(R 4 R 5 R 6 ) (wherein each of R 4 , R 5  and R 6  which are independent of one another, is an alkyl group or an aryl group), Z is an alkyl group, an aryl group, an oxygen atom, a sulfur atom, a carbonyl group, —(CO)—O—, —O—(CO)—, —NH—, —NH—CO—, —CO—NH—, —OSi(R 7 R 8 )— (wherein each of R 7  and R 8  which are independent of each other, is an alkyl group or aryl group) or a group of the formula —(R 9 R 10 )SiO— (wherein each of R 9  and R 10  which are independent of each other, is an alkyl group or an aryl group), each of R 1  and R 2  is an alkyl group or an aryl group, and R 3  is a spacer.

The present invention relates to 1,2-dioxetane derivatives. The1,2-dioxetane derivatives of the present invention are compounds whichare capable of inducing chemiluminescence and can be used, for example,as substrates for immunoassay.

Heretofore, various 1,2-dioxetane derivatives have been synthesized, andit is known that compounds having a spiroadamantyl group bonded at the3-position, are useful as chemiluminescent substrates (see, for example,JP-B-5-21918, and JP-B-5-45590) Further, as produced by the presentinventors, various compounds are known (see, for example, JP-A-8-245615,JP-A-8-169885, JP-A-8-165287 and JP-A-2002-338576). These 1,2-dioxetanederivatives have an enzyme recognition site such as a phosphate estergroup and will be triggered by an enzyme to produce luminescence. Aseries of such compounds are useful for a method wherein the activitiesof an enzyme labeled on an antigen or antibody adsorbed on a solid phaseafter an immunoreaction, are detected by a chemiluminescent reaction. Insuch a method, a 1,2-dioxetane solution is added to the measuringsystem, whereby even in a case no enzyme is present, luminescence by aheat or by a non-enzymatic decomposition reaction of e.g. a trace amountof impurities, will be detected, which leads to a rise of thebackground. Such a rise of the background is substantially influentialto the detection sensitivity and thus is problematic.

Therefore, various compounds have been synthesized (see, for example,Japanese Patent 2,572,171, JP-A-08-502968 and JP-A-2002-508654), but thelow stability of such dioxetane compounds themselves has still remainedas a problem to be solved.

As mentioned above, various studies have been made with respect to1,2-dioxetane derivatives, and various compounds have been produced.However, for such compounds to be useful in the fields of e.g. clinicaltests, the compounds themselves are required to be stable and easy tohandle and to have a performance to provide a low background formeasurement to present high sensitivity. Accordingly, it has beendesired to develop a compound superior to conventional compounds.

Under these circumstances, the present inventors have conducted anextensive study to develop a compound which is superior to conventionalcompounds and as a result, have succeeded in synthesizing a1,2-dioxetane derivative which has a stable structure and is able toreduce the background for the measurement by e.g. an immunoassay andwhich can be labeled to an organic compound or a biological molecule.The present invention has been accomplished on the basis of thisdiscovery.

Namely, the present invention provides a 1,2-dioxetane derivative of theformula (I):

wherein Ar is an aryl group which may have an alkyl group, an arylgroup, a halogen atom, an alkoxyl group, a carboxyl group, a formylgroup, an alkyl ester, an aryl ester, an alkylketone, an arylketone or ahetero ring bonded thereto, X is a substituent capable of labeling anorganic compound or a biological molecule, or an ester, Y is a hydrogenatom, an acyl group or a group of the formula —Si(R₄R₅R₆) (wherein eachof R₄, R₅ and R₆ which are independent of one another, is an alkyl groupor an aryl group), Z is an alkyl group, an aryl group, an oxygen atom, asulfur atom, a carbonyl group, —(CO)—O—, —O—(CO)—, —NH—, —NH—CO—,—CO—NH—, —OSi(R₇R₈)— (wherein each of R₇ and R₈ which are independent ofeach other, is an alkyl group or aryl group) or a group of the formula—(R₉R₁₀)SiO— (wherein each of R₉ and R₁₀ which are independent of eachother, is an alkyl group or an aryl group), each of R₁ and R₂ is analkyl group or an aryl group, and R₃ is a spacer.

Further, the present invention provides a 1,2-dioxetane derivative ofthe formula (III):

wherein Ar is an aryl group which may have an alkyl group, an arylgroup, a halogen atom, an alkoxyl group, a carboxyl group, a formylgroup, an alkyl ester, an aryl ester, an alkylketone, an arylketone or ahetero ring bonded thereto, X is a substituent capable of labeling anorganic compound or a biological molecule, or an ester, V is a carbonylgroup or a group of the formula —Si(R₁₅R₁₆)— (wherein each of R₁₅ andR₁₆ which are independent of each other, is an alkyl group or arylgroup), each of R₁₁ and R₁₂ which are independent of each other, is ahydrogen atom, an alkyl group or an aryl group, or R₁₁ and R₁₂ maytogether form a cyclic or polycyclic organic ring group spiro-bonded tothe dioxetane ring, R₁₃ is an alkyl group or an aryl group, or R₁₃ andR₁₁, or R₁₃ and R₁₂, may together form a condensed ring containing thedioxetane ring and a hetero atom, and R₁₄ is a spacer.

Still further, the present invention provides a chemiluminescent reagentwhich contains the above 1,2-dioxetane derivative. Still further, thepresent invention provides an immunoassay reagent wherein the above1,2-dioxetane derivative is bonded to a substance having a specificaffinity via a part of its X or W.

In the accompanying drawing,

FIG. 1 is a graph showing a luminescence curve obtained in Example 38.

Now, the present invention will be described in detail with reference tothe preferred embodiments.

In this specification, “an alkyl group” means a C₁₋₂₀ straight chain orbranched alkyl group which may have a substituent, and it may, forexample, be a straight chain group such as methyl, ethyl, propyl, butyl,pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl,tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl oricosanyl, or a group in which such an alkyl group is branched. Thesubstituent which such an alkyl group may have, is, for example, ahydroxyl group, an alkoxyl group or an aryl group.

In this specification, “an alkoxyl group” may, for example, be onehaving from 1 to 5 C₁₋₂₀ alkoxyl groups bonded in a straight chain formor in a branched form, such as methoxy, ethoxy, propoxy, butoxy,pentyloxy, hexyloxy, methoxyethoxy, methoxypropoxy, ethoxyethoxy,ethoxypropoxy or methoxyethoxyethoxy. In this specification, “an arylgroup” may, for example, be a C₆₋₂₀ aromatic hydrocarbon group such asphenyl or naphthyl, or a heteroaryl group having from 1 to 5 nitrogenatoms, oxygen atoms or sulfur atoms in a ring, such as furyl, thienyl orpyridyl.

In this specification, “an acyl group” may, for example, be a formylgroup, an acetyl group, a succinyl group, a benzoyl group, a 1-naphthoylgroup or a 2-naphthoyl group. Further, in this specification, “a cyclicorganic ring group” is a C₅₋₁₀ cyclic alkylene such as cyclohexyl orcycloheptyl, and “a polycyclic organic ring group” is a C₆₋₃₀ polycyclicalkylene which may optionally be substituted by from 1 to 10 groupsindependently selected from C₁₋₁₀ alkyl, C₁₋₁₀ alkoxyl, halogen andhalo-C₁₋₁₀ alkyl, such as an adamantyl group or a bicyclo[2.2.1]heptylgroup. Further, a halogen atom, an alkyl group, an aryl group, a cyanogroup, an amide group, an alkoxyl group or a carboxyl group may bebonded to optional carbon of such a polycyclic organic ring group.

Further, in this specification, “an hetero ring” may, for example, befuran, thiophene, pyrrol, oxazole, isooxazole, thiazole, isothiazole,imidazole, pyrazole, furazane, pyran, pyridine, pyridadine, pyrimidineor pyradine. “A halogen atom” may, for example, be fluorine, chlorine orbromine.

X is a substituent capable of labeling an organic compound or abiological molecule, or an ester. The substituent capable of labeling anorganic compound or a biological molecule, may, for example, be acarboxyl group, a succinimidoxy substituent, an acid chloride, an aminogroup, a maleimide group or a succinimidoxycarbonyl group, and the estermay, for example, be a C₁₋₆ alkyl ester. Each of R₃ and R₁₄ is a spacerand may, for example, be —(CH₂)_(n)— or —(CH₂)_(n)NH— (wherein n is aninteger of from 1 to 20).

Further, in the formula (III), the case wherein R₁₃ and R₁₁, or R₁₃ andR₁₂, together form a condensed ring containing the dioxetane ring and ahetero atom, may, for example, be a condensed ring of the dioxetane ringand a furan ring, or a condensed ring of the dioxetane ring and a pyranring.

Among those of the formula (I), preferred is a 1,2-dioxetane derivativeof the formula (II):

wherein Y is the same as Y in the formula (I), n is an integer of from 1to 20, W is a hydrogen atom, an alkyl group or a succinimidosubstituent, and U is a hydrogen atom, an alkyl group, an aryl group, ahalogen atom, an alkoxyl group, a carboxyl group, a formyl group, analkyl ester, an aryl ester, an alkylketone, an arylketone or a heteroring. More preferably, U is a hydrogen atom, n is from 1 to 15, W is ahydrogen atom, a C₁₋₆ alkyl group such as an ethyl group, or asuccinimido, and Y is a C₁₋₆ acyl group or —Si(R₄R₅R₆) (particularlyeach of R₄, R₅ and R₆ is a C₁₋₆ alkyl group).

Among those of the formula (III), preferred is a 1,2-dioxetanederivative of the formula (IV):

wherein n is an integer of from 1 to 20, W is a hydrogen atom, an alkylgroup or a succinimido substituent, and U is a hydrogen atom, an alkylgroup, an aryl group, a halogen atom, an alkoxyl group, a carboxylgroup, a formyl group, an alkyl ester, an aryl ester, an alkylketone, anarylketone or a hetero ring. More preferably, U is a hetero ring,particularly an isooxazole ring which may have a substituent such asCF₃, n is from 1 to 6, and W is a succinimido group, a hydrogen atom oran alkyl group.

The following process may, for example, be mentioned as a productionprocess wherein the compound of the formula (I) is a dihydrofuranderivative.

In the above formulae, R₃, X and Y are the same as R₃, X and Y asdefined in the above formula (I). Each of R₁₇ to R₂₆ which areindependent of one another, is a hydrogen atom, an alkyl group or anaryl group. R₂₇ is a halogen atom, a substituted sulfonyloxy group or ahydroxyl group. R₂₈ is an alkyl group.

Step 1: In this step, a compound of the above formula (1) is reactedwith a compound of the above formula (2) to produce a compound of theabove formula (3). This reaction can be carried out by a so-calledWilliamson synthesis which is well known to those skilled in the art.Here, when the substituent R₂₇ of the compound of the above formula (1)is a halogen atom or a substituted sulfonyloxy group, it may be directlysubjected to the reaction, and when R₂₇ is a hydroxyl group, it isfirstly converted to a sulfonyloxy group by e.g. a tosyl halide in thereaction system, and then subjected to the reaction, to accomplish thisstep.

Step 2: In this step, the compound of the above formula (3) is subjectedto a protective group-removing reaction to produce a compound of theabove formula (4). The protective group-removing reaction in this stepcan be carried out by means of an acid. As such an acid, hydrochloricacid may, for example, be employed, and in such a case, as a solvent, anether such as THF may be employed.

Step 3: In this step, one of alcoholic hydroxyl groups in the compoundof the above formula (4) is protected to produce a compound of the aboveformula (5). The protecting reaction in this step can be carried out byusing 3,4-dihydro-2H-pyran. In such a case, as a solvent, a halogenatedhydrocarbon such as dichloromethane may be employed. Further, as acatalyst, PPTS (pyridinium p-toluene sulfonate) may be employed, wherebythe desired product can be obtained efficiently.

Step 4: In this step, the compound of the above formula (5) is oxidizedto produce a compound of the above formula (6). The oxidation in thisstep can be carried out by using a chromium oxidizing agent or anactivating agent. Such a chromium oxidizing agent may, for example, bepyridinium chlorochromate (PCC) or pyridinium dichlorochromate (PDC). Insuch a case, as a solvent, a halogenated hydrocarbon such asdichloromethane may be employed. Whereas, when the above-mentionedactivating agent is to be used, the reaction may be carried out in acombination with a solvent, such as a Py.SO₃/triethylamine/DMSO systemor an Ac₂O/DMSO system.

Step 5: In this step, the compound of the above formula (6) is subjectedto ring closure to produce a compound of the above formula (7). Thereaction is carried out by using a base such as a lithium salt of asecondary amine such as lithium diisopropylamide, or t-butoxy potassium.As a solvent, an organic solvent such as THF or DMSO may be employed,and it is preferred to carry out the reaction from 0° C. to roomtemperature for from 1 to 5 hours.

Step 6: In this step, the compound of the above formula (7) is subjectedto a protective group-removing reaction to produce a compound of theabove formula (8). The group-removing reaction in this step can becarried out by using an acid. As such an acid, hydrochloric acid may,for example, be employed, and in such a case, as a solvent, an alcoholsuch as methanol can be used.

Step 7: In this step, the compound of the above formula (8) is reactedwith a compound having a R₃X substituent to produce a compound of theabove formula (9). This reaction can be accomplished by a so-calledWilliamson synthesis which is well known to those skilled in the art.

Step 8: In this step, the compound of the above formula (9) isdehydrated to produce a compound of the above formula (10). For thereaction, thionyl chloride may be reacted in the presence of a base suchas pyridine, or an acid such as phosphoric acid or p-toluene sulfonicacid may be used as a catalyst. As a solvent, a halogenated hydrocarbonsuch as dichloromethane, or an aromatic hydrocarbon such as toluene, maybe employed, and the solvent may suitably be selected for use dependingupon the reagent to be reacted.

Step 9: In this step, the compound of the above formula (10) issubjected to a protective group-removing reaction to produce a compoundof the above formula (11). In the case of a compound represented by amethoxy group or a benzyloxy group, this reaction can be carried out bya method well known to those skilled in the art, i.e. by reacting itwith an anion of an alkylthiol, or by subjecting it to a hydrogenationreaction. Either reaction may be selected for use depending upon thegroup to be removed.

Step 10: In this step, a compound which can be removed in the presenceof fluorine ions or under an alkaline condition, is introduced to thephenolic hydroxyl group of the compound of the above formula (11) toproduce a compound of the above formula (12). In order to form a grouprepresented by an alkyl ester, an aryl ester or —OSi(R₄R₅R₆) (whereineach of R₄, R₅ and R₆ which are independent of one another, is an alkylgroup or an aryl group), the corresponding acid anhydride or halogenatedsilane compound is reacted to produce the compound of the above formula(12).

Step 11: In this step, the compound of the above formula (12) is reactedwith singlet oxygen to produce a 1,2-dioxetane derivative of the aboveformula (13). The reaction with singlet oxygen can be accomplished bycarrying out visible light irradiation in an oxygen atmosphere in theco-existence of a photosensitizer such as Methylene Blue, Rose Bengaleor tetraphenyl porphine (TPP). Here, as a solvent, a halogenatedhydrocarbon such as dichloromethane, dichloroethane or carbontetrachloride, or an alcohol such as methanol or ethanol, may beemployed. Further, the reaction is preferably carried out at atemperature of from −80° C. to room temperature.

The compound of the above formula (III) may be produced, for example, byreacting an acid anhydride to a compound obtained by the methoddisclosed in e.g. JP-A-2002-338576.

The 1,2-dioxetane derivative of the above formula (I) or (III) of thepresent invention will be decomposed into a carbonyl compoundaccompanying chemiluminescence in the presence of fluorine ions or underan alkaline condition. Accordingly, such a derivative can be used as achemiluminescent reagent and can be used, for example, in animmunoassay, a chemical detecting method, a nucleotide probe method,etc.

Particularly, the 1,2-dioxetane derivative of the formula (I) or (III)of the present invention can be used as an immunoassay reagent bybonding it to a substance having a specific affinity via a part of its Xor W. An immunoassay employing such an immunoassay reagent may, forexample, be carried out by a step of mixing the immunoassay reagent ofthe present invention and a test sample containing a substance to bedetected and reacting them for a prescribed period of time to bond thesubstance to be detected in the sample with the substance having aspecific affinity thereto, and a step of obtaining the amount of thesubstance having the specific affinity, which was bonded or not bonded.Since the portion corresponding to the 1,2-dioxetane derivativeconstituting the immunoassay reagent of the present invention willdecompose accompanying a chemiluminescence in the presence of fluorineions or under an alkaline condition, the above-mentioned step ofobtaining the amount of the substance having a specific affinity, whichwas bonded or not bonded, can be carried out by measuring the intensityof the luminescence. The intensity of the luminescence in such a case,increases in proportion to the amount of the portion corresponding tothe 1,2-dioxetane derivative.

Substances to be detected in the above immunoassay include, for example,hormones such as hCG, TSH and LH, cancer-related substances such as AFPor CEA, viral antigens and antibodies, such as HIV or HTLV-I, andnucleic acids (DNA, RNA). Further, the substance having a specificaffinity, constituting the immunoassay reagent of the present invention,is one having a specific affinity to the substance to be detected, andit may, for example, be an antibody or a receptor.

Now, the present invention will be described in further detail withreference to Examples. However, it should be understood that the presentinvention is by no means restricted to such specific Examples.

EXAMPLE 1

In a nitrogen atmosphere at 0° C., to a DMF (100 mL) having potassiumcarbonate (73.2 g, 529.6 mmol, 3 eq.) suspended, dimethyl1,3-acetondicarboxylate (compound (1)) (30.9 g, 177.4 mmol) dissolved inDMF (25 mL) was dropwise added over a period of 12 minutes, and then,methyl iodide (33 mL, 530.1 mmol, 3 eq.) dissolved in DMF (20 mL) wasdropwise added over a period of 40 minutes. The reaction solution wasgradually returned to room temperature and stirred, and DMF (40 mL+50mL) was added, followed by stirring for one day. This reaction solutionwas put into water and extracted with ethyl acetate. The aqueous layerwas again extracted with ethyl acetate, and the extract was put togetherwith the previous organic layer, followed by washing with a saturatedsodium chloride aqueous solution. The organic layer was dried overanhydrous magnesium sulfate and concentrated to obtain the dimethyl1,3-dimethyl-2-oxo-1,3-propanedicarboxylate (compound (2)) as a yellowoil (39.1 g). This product was used for the subsequent reaction withoutpurification.

EXAMPLE 2

In a nitrogen atmosphere at 0° C., to a THF (100 mL) solution having 60%sodium hydride (17.9 g, 447.0 mmol, 2.6 eq.) suspended, the crudeproduct (39.1 g) of dimethyl 1,3-dimethyl-2-oxo-1,3-propanedicarboxylate(compound (2)) dissolved in THF (50 mL), was dropwise added over aperiod of 45 minutes, followed by stirring for 30 minutes. Then, methyliodide (33 mL, 530.1 mmol, 3 eq.) dissolved in THF (50 mL) was dropwiseadded over a period of 50 minutes, and the solution was graduallyreturned to room temperature and stirred for one day. This reactionsolution was put into water and extracted with ethyl acetate. Theaqueous layer was again extracted with ethyl acetate, and the extractwas put together with the previous organic layer, followed by washingwith a saturated sodium thiosulfate aqueous solution and a saturatedsodium chloride aqueous solution. The organic layer was dried overanhydrous magnesium sulfate and concentrated to obtain the desireddimethyl 1,1,3,3-tetramethyl-2-oxo-1,3-propanedicarboxylate (compound(3)) as a yellow oil (42.9 g). This product was used for the subsequentreaction without purification.

EXAMPLE 3

In a nitrogen atmosphere at 0° C., to a THF (150 mL) solution havinglithium aluminum hydride (10.2 g, 268.2 mmol, 1.5 eq.) suspended, thecrude product (42.9 g) of dimethyl1,1,3,3-tetramethyl-2-oxo-1,3-propanedicarboxylate (compound (3))dissolved in THF (50 mL), was dropwise added, and the solution wasgradually returned to room temperature and stirred for one day. To thisreaction solution, water (10 mL) dissolved in THF (10 mL) was added forquenching. This reaction solution was put into a 6N hydrochloric acidaqueous solution and extracted with ethyl acetate. The aqueous layer wasagain extracted with ethyl acetate, and the extract was put togetherwith the previous organic layer, followed by washing with a saturatedsodium chloride aqueous solution. The organic layer was dried overanhydrous magnesium sulfate and concentrated to obtain a residue as ayellow solid (28.7 g). This residue was rinsed with hexane to obtain ayellow solid (19.2 g, 109.2 mmol, 61.6%) of the desired2,2,4,4-tetramethylpentan-1,3,5-triol (compound (4)) and a concentrate(8.98 g) of the filtrate. The concentrate of the filtrate was subjectedto silica gel column chromatography with a developing solvent (ethylacetate:hexane=1:3). As a result, a yellow solid (2.14 g, 12.13 mmol,6.8%) was further obtained (the total yield of the compound (4): 68.4%).

Colorless needle crystal (mp. 61.1 to 61.5° C.)

¹H-NMR (400 MHz, CDCl₃): δ_(H) 1.00 (s, 6H). 1.09 (s, 6H), 2.95 (br,2H), 3.47 (d, J=10, 6 Hz, 2H). 3.52 (d, J=10, 6 Hz, 2H), 3.64 (s, 12),4.25 (br, 1H) ppm

¹³C-NMR (125 MHz, CDCl₃): δ_(C) 20.3, 24.7, 40.3, 75.4, 85.7 ppm

IR (KBr): 3354, 2954, 2878, 1028 cm⁻¹

MASS (EI, 70 ev, m/z, %): 176 (M⁺, trace), 128 (8), 103 (38), 97 (35),85 (24), 73 (36) 58 (2) 54 (100).

EXAMPLE 4

At room temperature, to a dichloromethane (200 mL) solution having2,2,4,4-tetramethylpentan-1,3,5-triol (compound (4)) (24.7 g, 139.9mmol) dissolved, acetone dimethylacetal (18 mL, 146.4 mmol, 1.1 eq.) wasadded, and then pyridinium p-toluene sulfonate (3.62 g, 13.99 mmol, 0.1eq.) was added, followed by stirring for one day. This reaction solutionwas put into a saturated sodium hydrogencarbonate aqueous solution andextracted with ethyl acetate. The aqueous layer was again extracted withethyl acetate, and the extract was put together with the previousorganic layer, followed by washing with a saturated sodium chlorideaqueous solution. The organic layer was dried over anhydrous magnesiumsulfate and concentrated to obtain a residue as a yellow oil (31.2 g).This residue was subjected to silica gel column chromatography with adeveloping solvent (ethyl acetate:hexane=1:4). As a result, the desired2-methyl-2-(2,2,5,5-tetramethyl-[1,3]dioxane-4-yl)-propan-1-ol (compound(5)) was obtained as a colorless oil (27.3 g, 126.3 mmol, 90.3%).

¹H-NMR (400 MHz, CDCl₃): δ_(H) 0.89 (s, 3H), 1.02 (s, 3H), 1.02 (s, 3H),1.21 (s, 3H), 1.42 (s, 3H), 1.42 (s, 3H), 3.01 (d, J=5.2 Hz, 1H), 3.11(d, J=11.5 Hz, 1H), 3.33 (d d, J=10.7 and 5.2 Hz, 1H), 3.54 (d, J=11.5Hz, 1H), 3.54 (dd, J=10.7 and 5.2 Hz, 1H), 3.59 (s, 1H) ppm

¹³C-NMR (100 MHz, CDCl₃): δ_(C) 18.7, 20.3, 21.2, 24.1, 24.5, 29.0,35.4, 40.2, 73.0, 74.4, 83.2, 98.5 ppm

IR (liquid film): 2994, 2858, 1601, 1462, 1264, 1044 cm⁻¹.

EXAMPLE 5

In a nitrogen atmosphere at 0° C., to a THF (30 mL) solution having 60%sodium hydride (968 mg, 24.00 mmol, 1.09 eq.) suspended, a THF (20 mL)solution having2-methyl-2-(2,2,5,5-tetramethyl-[1,3]dioxane-4-yl)-propan-1-ol (compound(5)) (4.76 g, 21.98 mmol) dissolved, was dropwise added over a period of15 minutes and 3-methoxybenzyl chloride (3.3 mL, 22.73 mmol, 1.03 eq.)was added. Then, TMF (5 mL) was added, and the solution was graduallyreturned to room temperature and heated at 50° C. and stirred for twohours. To this reaction solution, water was put, followed by extractionwith a saturated ammonium chloride aqueous solution and ethyl acetate.The aqueous layer was again extracted with ethyl acetate, and theextract was put together with the previous organic layer, followed bywashing with a saturated sodium chloride aqueous solution. The organiclayer was dried over anhydrous magnesium sulfate and concentrated toobtain a residue as a yellow oil (7.67 g). This residue was subjected tosilica gel column chromatography with a developing solvent (ethylacetate:hexane=1:10). As a result, the desired4-[2-(3-methoxybenziloxy)-1,1-dimethylethyl]-2,2,5,5-tetramethyl-[1,3]dioxane(compound (6)) was obtained as a colorless oil (6.62 g, 19.68 mmol,89.6%).

¹H-NMR (400 MHz, CDCl₃): δ_(H) 0.88 (s, 3H), 0.95 (s, 3H), 1.05 (s, 3H),1.14 (s, 3H), 1.34 (s, 3H), 1.37 (s, 3H), 3.01 (d, J=8.6 Hz, 1H), 3.08(d, J=11.5 Hz, 1H), 3.37 (d, J=8.6 Hz, 1H), 3.51 (d, J=11.5 Hz, 1H),3.65 (s, 1H), 3.81 (s, 3H), 4.42 (d, J=12.9 Hz, 1H), 4.47 (d, J=12.9 Hz,1H), 6.82 (d with fine coupling, J=8.1 Hz, 1H). 6.90–6.91 (m, 2H), 7.25(t, J=8.1 Hz, 1H) ppm

¹³C-NMR (100 MHz, CDCl₃): δ_(C) 19.1, 21.0, 21.9, 23.4, 24.3, 35.4,40.5, 55.1, 72.9, 74.7, 78.5, 98.4, 112.7, 119.5, 129.1, 140.5, 159.5ppm.

IR (liquid film): 3441, 2990, 2954, 2873, 1164, 1010, 938 cm⁻¹

MASS (EI, 70 ev, m/z, %): 336 (M⁺, 8), 321 (6), 278 (9), 222 (55), 194(5), 137 (36), 121 (100 ), 97 (12), 58 (55).

EXAMPLE 6

At room temperature, a THF (120 mL) solution having4-[2-(3-methoxybenziloxy)-1,1-dimethylethyl]-2,2,5,5-tetramethyl-[1,3]dioxane(compound (6)) (15.6 g, 46.34 mmol) dissolved, a 3N hydrochloric acidaqueous solution (15 mL) was added, and the mixture was refluxed at 80°C. for 6 hours and 40 minutes. The reaction solution was put into asaturated sodium hydrogencarbonate aqueous solution and extracted withethyl acetate. The aqueous layer was again extracted with ethyl acetate,and the extract was put together with the previous organic layer,followed by washing with a saturated sodium chloride aqueous solution.The organic layer was dried over anhydrous magnesium sulfate andconcentrated to obtain a residue as a yellow oil (14.2 g). This residuewas subjected to silica gel column chromatography with a developingsolvent (ethyl acetate:hexane=1:7). As a result, the desired5-(3-methoxybenzyloxy)-2,2,4,4-tetramethylpentan-1,3-diol (compound (7))as a colorless oil (11.5 g, 38.68 mmol, 83.5%).

¹H-NMR (400 MHz, CDCl₃): δ_(H) 0.98 (s, 3H), 1.02 (s, 3H), 1.06 (s, 3H),1.11 (s, 3H), 3.33 (s, 2H), 3.33–3.59 (m, 4H), 3.82 (s, 3H), 4.26 (br-s,1H), 4.49 (s, 2H), 6.84–6.89 (m, 3H), 7.25–7.29 (m, 1H) ppm

¹³C-NMR (125 MHz, CDCl₃): δ_(C) 20.1, 21.1, 24.7, 25.0, 40.4, 40.4,55.1, 73.6, 75.4, 83.0, 85.3, 112.9, 113.3, 120.0, 129.5, 139.0, 159.7ppm

IR (liquid film): 3415, 2957, 1600, 1266, 1155, 1079, 782 cm⁻¹

MASS (EI, 70 ev, m/z, %): 296 (M+, 19), 222 (8), 138 (94), 121 (100),109 (8); 73 (10).

EXAMPLE 7

At room temperature, to a dichloromethane (20 mL) solution having5-(3-methoxybenzyloxy)-2,2,4,4-tetramethylpentan-1,3-diol (compound (7))(1.87 g, 6.326 mmol) dissolved, 3,4-dihydro-2H-pyran (0.7 mL, 7.672mmol, 1.21 eq.) was added, and pyridinium p-toluene sulfonate (81.0 mg,0.3223 mmol, 0.05 eq.) was added, followed by stirring for one day. Thereaction solution was put into a saturated sodium hydrogencarbonateaqueous solution and extracted with ethyl acetate. The aqueous layer wasagain extracted with ethyl acetate, and the extract was put togetherwith the previous organic layer, followed by washing with a saturatedsodium chloride aqueous solution. The organic layer was dried overanhydrous magnesium sulfate and concentrated to obtain a residue as ayellow oil (2.53 g). This residue was subjected to silica gel columnchromatography with a developing solvent (ethyl acetate:hexane=1:5). Asa result, the desired1-(3-methoxybenzyloxy)-2,2,4,4-tetramethyl-5-(tetrahydropyran-2-yloxy)pentan-3-ol(compound (8)) was obtained as a colorless oil (1.74 g, 4.564 mmol,72.2%).

¹H-NMR (400 MHz, CDCl₃): δ_(H) 1.02 (s, 1.5H), 1.04 (s, 1.5H), 1.04 (s,1.5H), 1.05 (s, 1.5H), 1.06 (s, 1.5H), 1.09 (s, 1.5H), 1.10 (s, 1.5H),1.11 (s, 1.5H), 1.53–1.82 (m, 6H), 3.14–3.70 (m, 9H) 3.81 (s, 3H),3.81–3.86 (m, 1H), 4.48–4.58 (m, 3H), 6.81 (d with fine coupling, J=7.8Hz, 1H), 6.90 (m, 2H), 7.25 (t, J=7.8 Hz, 1H) ppm

¹³C-NMR (125 MHz, CDCl₃): δ_(C) 19.3, 19.6, 21.6, 21.9, 21.9, 22.0,24.4, 24.7, 24.9, 25.0, 25.3, 25.4, 30.5, 30.6, 40.4, 40.5, 40.8, 55.1,61.9, 62.4, 73.1, 77.7, 78.2, 80.4, 80.5, 80.6, 81.1, 99.0, 99.3, 112.7,112.9, 113.0, 119.6, 129.3, 129.3, 140.1, 140.2, 169.6 ppm

MASS (EI, 70 ev, m/z, %): 380 (M⁺, 3), 295 (29), 222 (13), 138 (60), 121(100), 85 (97).

EXAMPLE 8

At room temperature, to a dichloromethane (15 mL) solution havingpyridinium chlorochromate (1.31 g, 6.063 mmol, 1.58 eq.) and sellite(3.50 g) suspended, pyridine (0.46 mL, 6.335 mmol, 1.65 eq.) was added,and dichloromethane (5 mL) having1-(3-methoxybenzyloxy)-2,2,4,4-tetramethyl-5-(tetrahydropyran-2-yloxy)pentan-3-ol(compound (8)) (1.46 g, 3.837 mmol) dissolved, was dropwise added over aperiod of 5 minutes, followed by stirring for 4 days. To this reactionsolution, 2-propanol (4 mL) was added, followed by stirring for 30minutes, and then, diethyl ether (100 mL) was added, followed bystirring for 30 minutes. The solution was subjected to sellitefiltration, and the filtrate was concentrated to obtain a residue as agreen oil (1.46 g). The residue was subjected to silica gel columnchromatography with a developing solvent (ethyl acetate:hexane=1:7). Asa result, the desired1-(3-methoxybenzyloxy)-2,2,4,4-tetramethyl-5-(tetrahydropyran-2-yloxy)pentan-3-one(compound (9)) as a colorless oil (1.17 g, 3.099 mmol, 80.8%).

¹H-NMR (400 MHz, CDCl₃): δ_(H) 1.23 (s, 3H), 1.28 (s, 3H), 1.28 (s, 3H),1.32 (s, 3H), 1.46–1.70 (m, 6H), 3.46–3.83 (m, 6H), 3.80 (s, 3H), 4.47(s, 2H), 4.55 (t, J=3.2 Hz, 1H), 6.80 (d with fine coupling, J=8.1 Hz,1H), 6.86 (s, 1H), 6.87 (d, J=8.1 Hz, 1H), 7.23 (t, J=8.1 Hz, 1H) ppm

¹³C-NMR (100 MHz, CDCl₃): δ_(C) 19.3, 23.3, 23.5, 23.6, 23.7, 25.5,30.5, 50.1, 50.3, 55.1, 61.8, 63.0, 73.0, 76.0, 78.3, 98.9, 112.5,112.9, 119.5, 129.1, 140.1, 159.5, 215.9 ppm

IR (liquid film): 3441, 2990, 2954, 2873, 1164, 1010, 938 cm⁻¹

MASS (E1, 70 ev, m/z, %): 378 (M⁺, 3), 322 (4), 293 (21), 237 (2), 157(16), 138 (11), 121 (91), 85 (100).

EXAMPLE 9

In a nitrogen atmosphere at room temperature, to a THF (40 mL) solutionhaving diisopropylamine (6.5 mL, 46.38 mmol, 2.5 eq.) dissolved, an-butyllithium hexane solution (1.61 M solution, 28 mL, 46.08 mmol, 2.4eq.) was added, followed by stirring for 35 minutes. This reactionsolution was cooled to −78° C., and a THF (30 mL) solution having1-(3-methoxybenzyloxy)-2,2,4,4-tetramethyl-5-(tetrahydropyran-2-yloxy)pentan-3-one(compound (9)) (7.00 g, 18.50 mmol) dissolved, was dropwise added over aperiod of 30 minutes and stirred for two hours and 40 minutes. Water wasadded to this reaction solution for quenching. Then, this reactionsolution was put into a saturated ammonium chloride aqueous solution andextracted with ethyl acetate. The aqueous layer was again extracted withethyl acetate, and the extract was put together with the previousorganic layer, followed by washing with a saturated sodium chlorideaqueous solution. The organic layer was dried over anhydrous magnesiumsulfate and concentrated to obtain a residue as a yellow oil (7.56 g).This residue was subjected to silica gel column chromatography with adeveloping solvent (ethyl acetate:hexane=1:4). As a result, the desired3-hydroxy-2-(3-methoxybenzyloxy)-4,4-dimethyl-3-[1,1-dimethyl-2-(tetrahydropyran-2-yloxy)ethyl]tetrahydrofuran(compound (10)) was obtained as a colorless oil 10a (1.98 g, 5.230 mmol,28.3%), 10a+10b (2.06 g, 5.436 mmol, 29.4%), 10b (2.59 g, 6.850 mmol,37.0%), and the total (6.63 g, 17.62 mmol, 94.7%).

10a

¹H-NMR (400 MHz, CDCl₃): δ_(H) 1.03 (s, 3H), 1.29 (s, 3H), 1.35 (s, 3H),1.57 (s, 3H), 1.54–1.79 (m, 6H), 2.70 (d, J=10.0 Hz, 1H), 3.45–3.48 (m,1H), 3.76–3.80 (m, 2H), 3.80 (s, 3H). 3.87 (d, J=8.1 Hz, 1H), 4.13 (m,1H), 4.88 (br, 1H), 5.14 (s, 1H), 6.80 (d with fine coupling, J=8.1 Hz,1H), 7.11 (s, 1H), 7.12 (d, J=8.1 Hz, 1H), 7.20 (t, J=8.1 Hz, 1H) ppm.

¹³C-NMR (100 MHz, CDCl₃): δ_(C) 19.0, 25.3, 25.4, 30.3, 41.5, 47.9,55.2, 62.0, 78.4, 80.0, 88.0, 90.7, 98.7, 112.7, 113.6, 120.3, 128.5,142.0, 159.1 ppm

IR (liquid film): 3455, 2940, 2874, 1722, 1603, 1487, 1390, 1281, 1037,784 cm⁻¹

MASS (EI, 70 ev, m/z, %): 378 (M⁺, 2), 276 (21), 157 (33), 136 (100),126 (32), 107 (16), 85 (32), 55 (41).

10b

¹H-NMR (400 MHz, CDCl₃): δ_(H) 1.14 (s, 6H), 1.39 (s, 6H), 1.55–1.77 (m,6H), 3.45–3.56 (m, 2H), 3.60 (d, J=8.0 Hz, 1H), 3.81 (s, 3H), 3.78–3.80(m, 2H), 3.90 (d, J=8.0 Hz, 1H), 4.61 (s with fine coupling, 1H), 5.00(s, 1H), 6.80 (d with fine coupling, J=7.3 Hz, 1H), 7.12–7.26 (m, 3H)ppm

IR (liquid film): 3474, 2934, 1602, 1487, 1389, 1259, 1036, 779 cm⁻¹

MASS (EI, 70 ev, m/z, %): 378 (M⁺, 5), 276 (17), 157 (72), 136 (100),126 (31), 107 (14), 85 (54), 55 (36).

EXAMPLE 10

At room temperature, a methanol (10 mL) solution having3-hydroxy-2-(3-methoxyphenyl)-4,4-dimethyl-3-[1,1-dimethyl-2-(tetrahydropyran-2-yloxy)ethyl]tetrahydrofuran(compound (10)) (1.05 g, 2.774 mmol) dissolved, a 1N hydrochloric acidaqueous solution (one drop) was added. Then, a 1N hydrochloric acidaqueous solution (one drop) was further added, followed by stirring forone day. This reaction solution was put into a saturated sodiumhydrogencarbonate aqueous solution and extracted with ethyl acetate. Theaqueous layer was again extracted with ethyl acetate, and the extractwas put together with the previous organic layer, followed by washingwith a saturated sodium chloride aqueous solution. The organic layer wasdried over anhydrous magnesium sulfate and concentrated to obtain aresidue as a yellow oil (827 mg). This residue was subjected to silicagel column chromatography with a developing solvent (ethylacetate:hexane=1:2). As a result, the desired3-(2-hydroxy-1,1-dimethyl)-2-(3-methoxyphenyl)-4,4-dimethyltetrahydrofuran-3-ol(compound (11)) was obtained as a colorless oil (766 mg, 2.602 mmol,93.8%). Compound 11 was used for the subsequent reaction withoutpurifying the isomers.

Colorless granular crystals (mp. 102.0 to 102.2° C.)

¹H-NMR (400 MHz, CDCl₃): δ_(H) 0.79 (br-s, 3H), 1.01 (s, 3H), 1.25 (s,3H), 1.37 (s, 3H), 2.17 (t, J=5.0 Hz, 1H), 3.22 (dd, J=10.9 and 5.0 Hz,1H), 3.49–3.51 (m, 1H), 3.70 (d, J=8.1 Hz, 1H), 3.81 (s, 3H), 3.89 (d,J=8.1 Hz, 1H), 4.52 (br-s, 1H), 5.05 (s, 1H), 6.81 (d with finecoupling, J=8.0 Hz, 1H), 7.14 (s, 1H), 7.15 (d, J=8.0 Hz, 1H), 7.22 (t,J=8.0 Hz, 1H) ppm

IR (KBr): 3295, 2938, 2877, 1607, 1583, 1486, 1456, 1284, 1043, 779 cm⁻¹

MASS (EI, 70 ev, m/z, %): 294 (M⁺, 20), 276 (33), 236 (45), 136 (100),121 (32), 107 (23), 85 (43), 73 (12), 70 (29).

EXAMPLE 11

In a nitrogen atmosphere at 0° C., to a DMF (20 mL) solution having 60%sodium hydride (1.29 g, 32.25 mmol, 1.6 eq.) suspended, a DMF (40 mL)solution having3-(2-hydroxy-1,1-dimethyl)-2-(3-methoxyphenyl)-4,4-dimethyltetrahydrofuran-3-ol(compound (11)) (6.00 g, 20.38 mmol) dissolved, was dropwise added overa period of 20 minutes, and 5-bromo valric acid (5 mL, 31.33 mmol, 1.5eq.) was added, followed by stirring for 5 hours and 30 minutes. Thisreaction solution was put into a saturated ammonium chloride aqueoussolution and extracted with ethyl acetate. The aqueous layer was againextracted with ethyl acetate, and the extract was put together with theprevious organic layer, followed by washing with a saturated sodiumchloride aqueous solution. The organic layer was dried over anhydrousmagnesium sulfate and concentrated to obtain a residue as a yellow oil(10.5 g). This residue was subjected to silica gel column chromatographywith a developing solvent (ethyl acetate:hexane=1:4). As a result, thedesired3-(7-ethoxycarbonyl-1,1-dimethyl-3-oxaheptyl)-3-hydroxy-2-(3-methoxyphenyl)-4,4-dimethyltetrahydrofuran(compound (12)) was obtained as a colorless oil 12a (6.03 g, 14.26 mmol,70.0%), 12a+12b (1.68 g, 3.988 mmol, 19.6%), 12b (223 mg, 0.5285 mmol,2.6%), and the total (7.93 g, 18.77 mmol, 92.1%).

12a

¹H-NMR (400 MHz, CDCl₃): δ_(H) 0.79 (br-s, 3H), 1.06 (s, 3H), 1.19 (s,3H), 1.26 (t, J=7.1 Hz, 3H), 1.35 (s, 3H), 1.58–1.71 (m, 4H), 2.32 (t,J=7.1 Hz, 2H), 2.80 (d, J=9.3 Hz, 1H), 3.18–3.24 (br, 2H), 3.68 (d,J=8.1 Hz, 1H), 3.80 (s, 3H), 3.87 (d, J=8.1 Hz, 1H), 4.13 (q, J=7.1 Hz,2H), 4.88 (br, 1H), 5.04 (s, 1H), 6.80 (d with fine coupling, J=8.1 Hz,1H), 7.12 (s, 1H), 7.14 (d, J=8.1 Hz, 1H), 7.21 (t, J=8.1 Hz, 1H) ppm.

¹³C-NMR (125 MHz, CDCl₃): δ_(C) 14.1, 21.6, 23.3, 25.3, 28.8, 33.7,41.5, 47.7, 55.0, 60.1, 70.6, 80.0, 81.7, 88.4, 92.3, 112.8, 114.0,120.8, 128.4, 142.2, 159.0, 173.2 ppm

IR (liquid film): 3447, 2936, 2873, 1734, 1603, 1488, 1372, 1093, 784cm⁻¹

MASS (EI, 70 ev, m/z, %): 422 (M⁺, 8), 245 (100), 243 (53), 188 (9), 147(13), 136 (57), 107 (18) 101 (41), 83 (26), 55 (22).

12b

¹H-NMR (400 MHz, CDCl₃): δ_(H) 1.01 (s, 3H), 1.16 (s, 3H), 1.21 (s, 3H),1.25 (t, J=7.2 Hz, 3H), 1.37 (s, 3H), 1.32–1.37 (m, 2H), 1.49–1.52 (m,2H), 2.23 (t, J=7.5 Hz, 2H), 2.55 (dd, J=13.5 and 6.5 Hz, 1H), 2.82 (d,J=9.3 Hz, 1H), 2.94 (dt, J=13.5 and 6.5 Hz, 1H), 3.08 (d. J=9.3 Hz, 1H),3.44 (d, J=7.1 Hz, 1H), 3.81 (s, 3H), 4.12 (q, J=7.1 Hz, 2H), 4.12 (d,J=7.1 Hz, 1H), 6.83 (ddd, J=7.8 and 2.7 and 1.3 Hz, 1H), 7.10 (d withfine coupling, J=7.8 Hz, 1H), 7.16 (dd, J=2.7 and 1.3 Hz, 1H), 7.21 (t,J=7.8 Hz, 1H) ppm.

¹³C-NMR (100 MHz, CDCl₃): δ_(C) 14.3, 21.6, 23.7, 26.9, 28.7, 33.9,40.4, 48.4, 55.2, 60.2, 70.5, 81.0, 81.7, 83.9, 86.4, 113.5, 115.5,122.5, 128.3, 142.4, 159.0, 173.2 ppm

IR (liquid film): 3403, 2963, 2873, 1734, 1599, 1486, 1372, 1094, 778cm⁻¹

MASS (EI, 70 ev, m/z, %): 422 (M⁺, 21), 245 (24), 243 (21), 147 (28),136 (92), 107 (17), 101 (64), 55 (27).

EXAMPLE 12

In a nitrogen atmosphere at 0° C., to a dichloromethane (14 mL) solutionhaving3-(7-ethoxycarbonyl-1,1-dimethyl-3-oxaheptyl)-3-hydroxy-2-(3-methoxyphenyl)-4,4-dimethyltetrahydrofuran(compound (12)) (1.39 g, 3.298 mmol) dissolved, pyridine (2.42 mL, 32.99mmol, 10.0 eq.) was added, and thionyl chloride (0.3 mL, 4.113 mmol, 1.2eq.) was added. The solution was gradually returned to room temperature,followed by stirring for 7 hours and 35 minutes. This reaction solutionwas put into a saturated sodium hydrogencarbonate aqueous solution andextracted with ethyl acetate. The aqueous layer was again extracted withethyl acetate, and the extract was put together with the previousorganic layer, followed by washing with a saturated sodium chlorideaqueous solution. The organic layer was dried over anhydrous magnesiumsulfate and concentrated to obtain a residue as a yellow oil (1.29 g).This residue was subjected to silica gel column chromatography with adeveloping solvent (ethyl acetate:hexane=1:6). As a result, the desired4-(7-ethoxycarbonyl-1,1-dimethyl-3-oxaheptyl)-5-(3-methoxyphenyl)-3,3-dimethyl-2,3-dihydrofuran(compound (13)) was obtained as a colorless oil (1.04 g, 2.560 mmol,77.6%).

¹H-NMR (400 MHz, CDCl₃): δ_(H) 1.04 (s, 6H), 1.25 (t, J=7.1 Hz, 3H),1.31 (s, 6H), 1.54–1.57 (m, 2H), 1.66–1.70 (m, 2H), 2.32 (t, J=7.5 Hz,2H), 3.10 (s, 1H), 3.25 (t, J=6.2 Hz, 2H), 3.80 (s, 3H), 3.87 (s, 2H),3.80 (s, 3H), 3.87 (s, 2H), 4.12 (q, J=7.1 Hz, 2H), 6.85–6.86 (m, 2H),6.90 (dt, J=7.4 and 1.2 Hz, 1H), 7.21–7.25 (m, 1H) ppm.

¹³C-NMR (100 MHz, CDCl₃): δ_(C) 14.3, 21.9, 27.3, 27.4, 29.1, 34.1,37.0, 47.0, 55.2, 60.1, 70.4, 79.5, 83.0, 113.9, 115.1, 122.3, 122.3,128.7, 137.0, 151.0, 158.9, 173.5 ppm

IR (liquid film): 2956, 2866, 1735, 1596, 1465, 1370, 1048, 785 cm⁻¹

MASS (EI, 70 ev, m/z %): 404 (M⁺, 2), 258 (19), 245 (100), 243 (43), 135(20), 55 (6).

EXAMPLE 13

In a nitrogen atmosphere at 0° C., a DMF (4 mL) solution having 60%sodium hydride (465 mg, 11.63 mmol, 4.2 eq.) suspended, ethane thiol (1mL, 13.60 mmol, 4.8 eq.) was added. The solution was returned to roomtemperature and then a DMF (7 mL) solution having4-(7-ethoxycarbonyl-1,1-dimethyl-3-oxaheptyl)-5-(3-methoxyphenyl)-3,3-dimethyl-2,3-dihydrofuran(compound (13)) (1.13 g, 2.791 mmol) dissolved, was dropwise added overa period of 5 minutes, followed by refluxing for 11 hours. This reactionsolution was put into a saturated ammonium chloride aqueous solution andextracted with ethyl acetate. The aqueous layer was again extracted withethyl acetate, and the extract was put together with the previousorganic layer, followed by a saturated sodium chloride aqueous solution.The organic layer was dried over anhydrous magnesium sulfate andconcentrated to obtain a residue as a yellow oil (1.18 g). This residuewas subjected to silica gel column chromatography with a developingsolvent (ethyl acetate:hexane=1:1). As a result, the desired4-(7-carboxy-1,1-dimethyl-3-oxaheptyl)-5-(3-hydroxyphenyl)-3,3-dimethyl-2,3-dihydrofuran(compound (14)) was obtained as a colorless oil (607 mg, 1.674 mmol,60.0%).

¹H-NMR (400 MHz, CDCl₃): δ_(H) 1.03 (s, 6H), 1.30 (s, 6H), 1.56–1.63 (m,2H), 1.70–1.77 (m, 2H), 2.42 (t, J=7.1 Hz, 2H), 3.10 (s, 2H), 3.26 (t,J=5.9 Hz, 2H), 3.86 (s, 2H), 6.79 (d with fine coupling, J=2.6 Hz, 1H),6.86 (s, 1H), 6.85 (d, J=11.2 Hz, 1H), 7.17 (t, J=7.8 Hz, 1H) ppm.

¹³C-NMR (100 MHz, CDCl₃): δ_(C) 21.9, 27.3, 27.5, 28.9, 33.7, 37.1,47.0, 70.5, 79.5, 82.9, 115.3, 116.9, 122.0, 122.0, 128.9, 136.9, 150.7,155.2, 178.8 ppm

IR (liquid film): 3376, 2957, 2869, 1709, 1595, 1445, 1047, 787 cm⁻¹

MASS (EI, 70 ev, m/z, %): 362 (M⁺, 3), 244 (22), 231 (100), 229 (46),121 (37), 55 (10).

EXAMPLE 14

In a nitrogen atmosphere at room temperature, to a dichloromethane (20mL) solution having4-(7-carboxy-1,1-dimethyl-3-oxaheptyl)-5-(3-hydroxyphenyl)-3,3-dimethyl-2,3-dihydrofuran(compound (14)) (2.17 g, 6.000 mmol) dissolved, triethylamine (3 mL,21.55 mmol, 3.6 eq.) was added. Further, the temperature was lowered to0° C., and acetic anhydride (1 mL, 10.60 mmol, 1.8 eq.) was added. Thesolution was returned to room temperature and stirred for 8 hours and 10minutes. This reaction solution was put into a saturated sodium chlorideaqueous solution and extracted with ethyl acetate. The aqueous layer wasagain extracted with ethyl acetate, and the extract was put togetherwith the previous organic layer, followed by washing with a saturatedsodium chloride aqueous solution. The organic layer was dried overanhydrous magnesium sulfate and concentrated to obtain a residue as ayellow oil (2.66 g). This residue was subjected to silica gel columnchromatography with a developing solvent (ethyl acetate:hexane=1:4). Asa result, the desired5-(3-acetoxyphenyl)-4-(7-carboxy-1,1-dimethyl-3-oxaheptyl)-3,3-dimethyl-2,3-dihydrofuran(compound (15)) was obtained as a colorless oil (1.36 g, 3.335 mmol,55.6%).

¹H-NMR (400 MHz, CDCl₃): δ_(H) 1.05 (s, 6H), 1.30 (s, 6H), 1.54–1.61 (m,2H), 1.67–1.74 (m, 2H), 2.28 (s, 3H), 2.38 (t, J=7.3 Hz, 2H), 3.08 (s,2H), 3.24 (t, J=6.1 Hz, 2H), 3.86 (s, 2H), 7.05 (ddd, J=7.8 and 2.4 and1.2 Hz, 1H), 7.08 (t, J=1.2 Hz, 1H), 7.19 (dt, J=7.8 and 1.2 Hz, 1H),7.32 (t, J=7.8 Hz, 1H) ppm.

¹³C-NMR (125 MHz, CDCl₃): δ_(C) 21.1, 21.7, 27.3, 27.4, 28.9, 33.7,37.0, 47.1, 70.3, 79.5, 83.1, 121.2, 123.0, 123.2, 127.3, 128.7, 137.2,150.0, 150.1, 169.2, 179.2 ppm

IR (liquid film): 2957, 2868, 1767, 1708, 1603, 1583, 1367, 1204, 785,706 cm⁻¹

MASS (EI, 70 ev, m/z, %): 404 (M⁺, 1), 273 (100), 271 (47), 229 (13),163 (10), 121 (21).

EXAMPLE 15

In an oxygen atmosphere at 0° C., to a dichloromethane solution having5-(3-acetoxyphenyl-)-4-(7-carboxy-1,1-dimethyl-3-oxaheptyl)-3,3-dimethyl-2,3-dihydrofuran(compound (15)) (104 mg, 0.2666 mmol) dissolved, TPP (1.0 mg) was added,and the solution was irradiated by a 940 W sodium lamp for 30 minutes,stirred and concentrated to obtain a residue as a red oil (129 mg). Thisresidue was subjected to silica gel column chromatography with adeveloping solvent (ethyl acetate:hexane=1:2). As a result, the desired1-(3-acetoxyphenyl)-5-(7-carboxy-1,1-dimethyl-3-oxaheptyl)-4,4-dimethyl-2,6,7-trioxabicyclo[3.2.0]heptane(compound (16)) was obtained as a yellow oil (105 mg, 0.2412 mmol,94.0%).

¹H-NMR (400 MHz, CDCl₃): δ_(H) 0.87 (s, 3H), 1.14 (s, 3H), 1.15 (s, 3H),1.38 (s, 3H), 1.54–1.58 (m, 2H), 1.63–1.68 (m, 2H), 2.30 (s, 3H), 2.35(t, J=7.3 Hz, 2H), 3.24–3.32 (m, 4H), 3.82 (d, J=8.2 Hz, 1H), 4.58 (d,J=8.2 Hz, 1H), 7.14 (d with fine coupling, J=8.0 Hz, 1H), 7.36 (s withfine coupling, 1H), 7.41 (t, J=8.0 Hz, 1H), 7.51 (d, J=8.0 Hz, 1H), 9.79(br, 1H) ppm.

¹³C-NMR (125 MHz, CDCl₃): δ_(C) 17.7, 20.6, 21.1, 21.5, 22.2, 24.8,28.8, 33.6, 41.1, 45.6, 70.5, 75.9, 80.3, 105.0, 116.4, 122.0, 122.7,125.7, 128.9, 137.4, 150.3, 169.1, 179.5 ppm

IR (liquid film): 2956, 1767, 1709, 1487, 1370, 1206, 793, 700 cm⁻¹

MASS (EI, 70 ev, m/z, %): 404 (M⁺, 1), 273 (18), 163 (91), 121 (54), 101(100), 83 (30).

EXAMPLE 16

In a nitrogen atmosphere at room temperature, to a THF (2.5 mL) solutionhaving5-(3-acetoxyphenyl)-4-(7-carboxy-1,1-dimethyl-3-oxaheptyl)-3,3-dimethyl-2,3-dihydrofuran(compound (15)) (209 mg, 0.5167 mmol) dissolved, ethanol (48 mg, 1.042mmol, 2.0 eq.) and triphenyl phosphine (275 mg, 1.048 mmol, 2.0 eq.)were added, and further diethyl azodicarboxylate (185 mg, 1.062 mmol,2.1 eq.) dissolved in THF (0.5 mL) was added, followed by stirring for20 minutes. This reaction solution was put into a 1N hydrochloricaqueous solution and a saturated sodium chloride aqueous solution, andextracted with ethyl acetate. The aqueous layer was again extracted withethyl acetate, and the extract was put together with the previousorganic layer, followed by washing with a saturated sodiumhydrogencarbonate aqueous solution and a saturated sodium chlorideaqueous solution. The organic layer was dried over anhydrous magnesiumsulfate and concentrated to obtain a residue. This residue was subjectedto silica gel column chromatography with a developing solvent (ethylacetate:hexane=1:20). As a result, the desired5-(3-acetoxyphenyl)-4-(7-ethoxycarbonyl-1,1-dimethyl-3-oxaheptyl)-3,3-dimethyl-2,3-dihydrofuran(compound (17)) was obtained as a colorless oil (172 mg, 0.3976 mmol,77.0%).

¹H-NMR (400 MHz, CDCl₃): δ_(H) 1.04 (s, 6H), 1.25 (t, J=7.1 Hz, 3H),1.30 (s, 6H), 1.53–1.58 (m, 2H), 1.64–1.70 (m, 2H), 2.78 (s, 3H), 2.32(t, J=7.4 Hz, 2H), 3.07 (s, 2H), 3.23 (t, J=6.4 Hz, 2H), 3.86 (s, 2H),4.12 (q, J=7.2 Hz, 2H), 7.05 (ddd, J=7.9 and 2.5 and 1.2 Hz, 1H), 7.08(t, J=1.2 Hz, 1H), 7.18 (td, J=7.9 and 1.2 Hz, 1H), 7.32 (t, J=7.9 Hz,1H) ppm.

¹³C-NMR (125 MHz, CDCl₃): δ_(C) 14.2, 21.0, 21.9, 27.2, 27.3, 29.0,34.1, 37.0, 47.1, 60.1, 70.4, 79.4, 83.0, 121.2, 123.0, 123.2, 127.2,128.6, 137.2, 150.0, 169.0, 173.6 ppm

IR (liquid film): 2957, 2867, 1768, 1735, 1203 cm⁻¹

MASS (EI, 70 ev, m/z, %): 432 (M⁺, 1), 286 (33), 273 (100), 229 (14),163 (5), 149 (37), 129 (8), 121 (14), 101 (8).

EXAMPLE 17

In an oxygen atmosphere at 0° C., to a dichloromethane (7 mL) solutionhaving5-(3-acetoxyphenyl)-4-(7-ethoxycarbonyl-1,1-dimethyl-3-oxaheptyl)-3,3-dimethyl-2,3-dihydrofuran(compound (17)) (76 mg, 0.1750 mmol) dissolved, TPP (0.8 mg) was added,and the solution was irradiated by a 940 W sodium lamp for 45 minutes,stirred and concentrated to obtain a residue as a yellow oil. Thisresidue was subjected to silica gel column chromatography with adeveloping solvent (ethyl acetate:hexane=1:10). As a result, the desired1-(3-acetoxyphenyl)-5-(7-ethoxycarbonyl-1,1-dimethyl-3-oxaheptyl)-4,4-dimethyl-2,6,7-trioxabicyclo[3.2.0]heptane(compound (18)) was obtained as a yellow oil (78 mg, 0.1679 mmol,95.9%).

¹H-NMR (400 MHz, CDCl₃): δ_(H) 0.86 (s, 3H), 1.15 (s, 3H), 1.16 (s, 3H),1.25 (t, J=7.1 Hz, 3H), 1.38 (s, 3H), 1.52–1.55 (m, 2H), 1.61–1.66 (m,2H), 2.29 (t, J=7.7 Hz, 2H), 2.30 (s, 3H), 3.25 (dd, J=9.3 and 7.7 Hz,2H), 3.29 (t, J=6.2 Hz, 2H), 3.82 (d, J=8.2 Hz, 1H), 4.12 (q, J=7.1 Hz,2H), 4.58 (d, J=8.2 Hz, 1H), 7.14 (d with fine coupling, J=8.0 Hz, 1H),7.37 (s with fine coupling, 1H), 7.41 (t, J=8.0 Hz, 1H), 7.51 (d, J=8.0Hz, 1H) ppm.

¹³C-NMR (125 MHz, CDCl₃): δ_(C) 14.2, 21.1, 21.8, 22.3, 28.9, 34.0,41.1, 45.6, 60.2, 70.6, 75.9, 80.3, 105.0, 116.4, 121.9, 122.7, 125.7,128.9, 137.5, 150.4, 169.1, 173.6 ppm

IR (liquid film): 2979, 1767, 1733, 1487, 1370, 1205, 701 cm⁻¹

MASS (EI, 70 ev, m/z, %): 432 (M⁺, trace), 319 (15), 263 (35), 229 (20),163 (91), 154 (trace), 149 (10), 129 (100), 121 (33), 101 (36).

EXAMPLE 18

To a solution having sodium hydride (60% in mineral oil, 412 mg, 10.3mmol) suspended in anhydrous DMF (7 mL) in a nitrogen stream at 0° C.,3-hydroxy-3-(2-hydroxy-1,1-dimethylethyl)-2-(3-methoxyphenyl)-4,4-dimethyltetrahydrofuran(compound (11)) (1.51 g, 5.13 mmol) dissolved in anhydrous DMF (4 mL),was dropwise added, followed by stirring at 0° C. for 30 minutes and atroom temperature for 20 minutes. To this solution, ethyl11-iodoundecanoate (3.51 g, 10.3 mmol) dissolved in anhydrous DMF (3 mL)was added at 0° C., followed by stirring for 4 hours and then bystirring at room temperature overnight. The reaction mixture was putinto a saturated ammonium chloride aqueous solution and extracted withethyl acetate. The organic layer was washed with a saturated sodiumchloride aqueous solution, dried over anhydrous magnesium sulfate andthen concentrated. The concentrate was put into a silica gel column anddeveloped with a mixed solvent of hexane and ethyl acetate (4:1),whereby3-(13-ethoxycarbonyl-1,1-dimethyl-3-oxatridecan-1-yl)-2-(3-methoxyphenyl-4,4-dimethyltetrahydrofuran(compound (19)) was obtained as a colorless oil in an amount of 2.52 gin a yield of 97.0%.

¹H-NMR (400 Hz, CDCl₃) δ_(H) 0.78 (s, 3H), 1.01 (s, 3H), 1.20–1.45 (m,21H), 1.57–1.67 (m, 2H), 1.77–1.87 (m, 2H), 2.29 (t, J=7.6 Hz, 2H), 3.19(t, J=7.1 Hz, 2H), 3.42–3.65 (m, 1H), 3.70 (d, J=8.1 Hz, 1H), 3.81 (s,3H), 3.89 (d, J=8.1 Hz, 1H), 4.12 (q, J=7.2 Hz, 2H), 4.50–4.70 (m, 1H),5.04 (s, 1H), 6.81 (d with fine coupling, J=7.8 Hz, 1H), 7.12–7.17 (m,2H), 7.22 (t, J=7.8 Hz, 1H) ppm.

EXAMPLE 19

To a solution having3-(13-ethoxycarbonyl-1,1-dimethyl-3-oxatridecan-1-yl)-2-(3-methoxyphenyl-4,4-dimethyltetrahydrofuran(compound (19)) (1.21 g, 2.39 mmol) and pyridine (2.0 mL, 24.7 mmol)dissolved in anhydrous dichloromethane (12 mL) in a nitrogen stream andstirred at 0° C., thionyl chloride (0.25 mL, 3.43 mmol) was added,followed by stirring for 5 minutes and then by stirring at roomtemperature for one hour. The reaction mixture was put into a sodiumhydrogencarbonate aqueous solution and extracted with ethyl acetate. Theorganic layer was washed with a saturated sodium chloride aqueoussolution, dried over anhydrous magnesium sulfate and then concentrated.The concentrate was put into a silica gel column and developed with amixed solvent of hexane and ethyl acetate (10:1), whereby4-(13-ethoxycarbonyl-1,1-dimethyl-3-oxatridecan-1-yl)-5-(3-methoxyphenyl-3,3-dimethyl-2,3-dihydrofuran(compound (20)) was obtained as a colorless oil in an amount of 1.06 gin a yield of 90.8%.

¹H-NMR (400 Hz, CDCl₃) δ_(H) 1.04 (s, 6H), 1.23–1.34 (m, 21H), 1.46–1.55(m, 2H) 1.57–1.65 (m, 2H), 2.28 (t, J=7.6 Hz, 2H) 3.11 (s, 2H), 3.24 (t,J=6.6 Hz, 2H), 3.80 (s, 3H), 3.87 (s, 2H), 4.12 (q, J=7.1 Hz, 2H).6.83–6.88 (m, 2H), 6.91 (d with fine coupling, J=7.5 Hz, 1H), 7.22 (twith fine coupling, J=7.5 Hz, 1H) ppm.

EXAMPLE 20

To a solution having sodium hydride (60% in mineral oil, 704 mg, 17.6mmol) suspended in anhydrous DMF (30 mL) in a nitrogen stream at 0° C.,ethanethiol (1.5 mL, 20.3 mmol) was dropwise added, followed by stirringat room temperature for a few minutes. This solution was added to4-(13-ethoxycarbonyl-1,1-dimethyl-3-oxatridecan-1-yl)-5-(3-methoxyphenyl-3,3-dimethyl-2,3-dihydrofuran(compound (20)) in a nitrogen stream, followed by heating and stirringat 140° C. for 20 minutes and then at 150° C. for one hour. The reactionmixture was put into dilute hydrochloric acid and extracted with ethylacetate. The organic layer was washed with a saturated sodium chlorideaqueous solution, dried over anhydrous magnesium sulfate and thenconcentrated. The concentrate was put into a silica gel column anddeveloped with a mixed solvent of hexane and ethyl acetate (2:1),whereby4-(13-carboxy-1,1-dimethyl-3-oxatridecan-1-yl)-5-(3-hydroxyphenyl)-3,3-dimethyl-2,3-dihydrofuran(compound (21)) was obtained as a colorless oil in an amount of 1.579 gin a yield of 94.2%.

¹H-NMR (400 Hz, CDCl₃) δ_(H) 1.04 (s, 6H), 1.20–1.40 (m, 18H), 1.46–1.55(m, 2H), 1.58–1.68 (m, 2H), 2.35 (t, J=7.4 Hz, 2H), 3.12 (s, 2H), 3.25(t, J=6.6 Hz, 2H), 3.86 (s, 2H), 6.76 (d with fine coupling, J=8.0 Hz,1H), 6.79 (s with fine coupling, 1H), 6.88 (d, 1H), 7.17 (dd, J=8.0 and7.6 Hz, 1H) ppm.

EXAMPLE 21

To a solution having4-(13-carboxy-1,1-dimethyl-3-oxatridecan-1-yl)-5-(3-hydroxyphenyl)-3,3-dimethyl-2,3-dihydrofuran(compound (21)) (1.57 g, 3.52 mmol) and triethylamine (2.5 mL, 17.9mmol) dissolved in anhydrous dichloromethane (15 mL) and stirred in anitrogen atmosphere at 0° C., acetic anhydride (0.50 mL, 5.30 mmol) wasadded, followed by stirring for 1.5 hours. The reaction mixture was putinto dilute hydrochloric acid and extracted with ethyl acetate. Theorganic layer was washed with a saturated sodium chloride aqueoussolution, dried over anhydrous magnesium sulfate and then concentrated.The concentrate was put into a silica gel column and developed with amixed solvent of hexane and ethyl acetate (2:1), whereby5-(3-acetoxyphenyl)-4-(13-carboxy-1,1-dimethyl-3-oxatridecan-1-yl)-3,3-dimethyl-2,3-dihydrofurananhydride was obtained as a colorless oil in an amount of 1.353 g in ayield of 80.2%. Then,5-(3-acetoxyphenyl)-4-(13-carboxy-1,1-dimethyl-3-oxatridecan-1-yl)-3,3-dimethyl-2,3-dihydrofuran(compound (22)) was obtained in an amount of 126 mg in a yield of 7.3%.

¹H-NMR (400 Hz, CDCl₃) δ_(H) 1.03 (s, 6H), 1.20–1.37 (m, 18H), 1.46–1.55(m, 2H), 1.57–1.67 (m, 2H), 2.27 (s, 3H), 2.34 (t, J=7.5 Hz, 2H), 3.09(s, 2H), 3.23 (t, J=6.5 Hz, 2H), 3.86 (s, 2H), 7.05 (ddd, J=8.1 and 2.4and 1.1 Hz, 1H), 7.09 (s with fine coupling, 1H), 7.20 (d with finecoupling, J=7.7 Hz, 1H), 7.31 (dd, J=8.1 and 7.7 Hz, 1H) ppm.

EXAMPLE 22

5-(3-acetoxyphenyl)-4-(13-carboxy-1,1-dimethyl-3-oxatridecan-1-yl)-3,3-dimethyl-2,3-dihydrofuran(compound (22)) (125 mg, 0.256 mmol) and TPP (1 mg) were added todichloromethane (12 mL), and the mixture was irradiated with visiblelight by a 940 W sodium lamp in an oxygen atmosphere at 0° C. for 30minutes. The reaction mixture was concentrated, and the concentrate wasput into a silica gel column and developed with a mixed solvent ofhexane and ethyl acetate (10:1 to 5:1), whereby1-(3-acetoxyphenyl)-5-(13-carboxy-1,1-dimethyl-3-oxatridecan-1-yl)-4,4-dimethyl-2,6,7-trioxabicyclo[3.2.0]heptane(compound (23)) was obtained in an amount of 133 mg in a yield of 97.6%.

¹H-NMR (400 Hz, CDCl₃) δ_(H) 0.86 (s, 3H), 1.15 (s, 3H), 1.16 (s, 3H),1.20–1.40 (m, 12H), 1.39 (s, 3H), 1.43–1.52 (m, 2H), 1.58–1.67 (m, 2H),2.30 (s, 3H), 2.35 (t, J=7.5 Hz, 2H), 3.23 (s, 2H), 3.27 (t with finecoupling, J=6.6 Hz, 2H) 3.82 (d, J=8.1 Hz, 1H), 4.58 (d, J=8.1 Hz, 1H),7.14 (ddd, J=8.1 and 2.3 and 1.1 Hz, 1H), 7.37 (s with fine coupling,1H), 7.40 (dd, J=8.1 and 7.9 Hz, 1H), 7.51 (d, J=7.9 Hz, 1H) ppm.

EXAMPLE 23

To a solution having ethanol (34 mg, 0.74 mmol), triphenyl phosphine(204 mg, 0.78 mmol) and5-(3-acetoxyphenyl)-4-(13-carboxy-1,1-dimethyl-3-oxatridecan-1-yl)-3,3-dimethyl-2,3-dihydrofuran(compound (22)) (126 mg, 0.258 mmol) dissolved in anhydrous THF (1.0 mL)in a nitrogen stream at room temperature and stirred, diethylazodicarboxylate (135 mg, 0.77 mmol) dissolved in anhydrous THF (0.5mL), was added, followed by stirring for 20 minutes. After completion ofthe reaction, the reaction mixture was put into dilute hydrochloric acidand extracted with ethyl acetate. The organic layer was washed with asaturated sodium chloride aqueous solution, dried over anhydrousmagnesium sulfate and then concentrated. The concentrate was put into asilica gel column and developed with a mixed solvent of hexane and ethylacetate (20:1 to 10:1), whereby5-(3-acetoxyphenyl)-4-(13-ethoxycarbonyl-1,1-dimethyl-3-oxatridecan-1-yl)-3,3-dimethyl-2,3-dihydrofuran(compound (24)) was obtained as a colorless oil in an amount of 111 mgin a yield of 83.3%.

¹H-NMR (500 Hz, CDCl₃) δ_(H) 1.03 (s, 6H), 1.23–1.34 (m, 21H), 1.47–1.54(m, 2H), 1.57–1.65 (m, 2H), 2.28 (s, 3H), 2.28 (t, J=7.6 Hz, 2H), 3.08(s, 2H), 3.22 (t, J=6.5 Hz, 2H), 3.86 (s, 2H), 4.12 (q, J=7.1 Hz, 1H),7.05 (ddd, J=8.0 and 2.4 and 1.0 Hz, 1H), 7.09 (s with fine coupling,1H), 7.20 (d with fine coupling, J=7.6 Hz, 1H), 7.32 (dd, J=8.0 and 7.6Hz, 1H) ppm.

EXAMPLE 24

5-(3-acetoxyphenyl)-4-(13-ethoxycarbonyl-1,1-dimethyl-3-oxatridecan-1-yl)-3,3-dimethyl-2,3-dihydrofuran(compound (24)) (57.8 mg, 0.112 mmol) and TPP (0.6 mg) were added todichloromethane (6 mL), and the mixture was irradiated with visiblelight by a 940 W sodium lamp in an oxygen atmosphere at 0° C. for 30minutes. The reaction mixture was concentrated, and the concentrate wasput into a silica gel column and developed with a mixed solvent ofhexane and ethyl acetate (10:1 to 5:1), whereby1-(3-acetoxyphenyl)-5-(13-ethoxycarbonyl-1,1-dimethyl-3-oxatridecan-1-yl)-4,4-dimethyl-2,6,7-trioxabicyclo[3.2.0]heptane(compound (25)) was obtained as a colorless oil in an amount of 46 mg ina yield of 75.0%.

¹H-NMR (400 Hz, CDCl₃) δ_(H) 0.86 (s, 3H), 1.15 (s, 3H), 1.16 (s, 3H),1.22–1.38 (m, 15H), 1.39 (s, 3H), 1.44–1.52 (m, 2H), 1.57–1.65 (m, 2H),2.28 (t, J=7.6 Hz, 2H), 2.30 (s, 3H), 3.23 (s, 2H), 3.26 (t with finecoupling, J=6.6 Hz, 2H), 3.82 (d, J=8.1 Hz, 1H), 4.12 (q, J=7.2 Hz, 2H),4.58 (d, J=8.1 Hz, 1H), 7.14 (d with fine coupling, J=7.6 Hz, 1H), 7.37(s with fine coupling, 1H), 7.41 (dd, J=8.0 and 7.8 Hz, 1H), 7.52 (d,J=7.8 Hz, 1H) ppm.

EXAMPLE 25

In a nitrogen atmosphere at 0° C., to a DMF (10 mL) solution having4-(7-carboxy-1,1-dimethyl-3-oxaheptyl)-5-(3-hydroxyphenyl)-3,3-dimethyl-2,3-dihydrofuran(compound (14)) (500 mg, 1.379 mmol) dissolved, imidazole (282 mg, 4.142mmol, 3.0 eq.) was added, and further, t-butyldimethylchlorosilane (624mg, 4.140 mmol, 3.0 eq.) was added. The solution was returned to roomtemperature and stirred for two hours. This reaction solution was putinto a saturated sodium chloride aqueous solution and extracted withethyl acetate. The aqueous layer was again extracted with ethyl acetate,and the extract was put together with the previous organic layer,followed by washing with a saturated sodium chloride aqueous solution.The organic layer was dried over anhydrous magnesium sulfate andconcentrated to obtain a residue as a yellow oil (806 mg). The residuewas dissolved in methanol (15 mL) and cooled to 0° C. Water (5 mL)having potassium carbonate (380 mg, 2.749 mmol) dissolved therein, wasdropwise added thereto, followed by stirring for 30 minutes. Thisreaction solution was put into a saturated sodium chloride aqueoussolution and extracted with ethyl acetate. The aqueous layer was againextracted with ethyl acetate, and the extract was put together with theprevious organic layer, followed by washing with a saturated sodiumchloride aqueous solution. The organic layer was dried over anhydrousmagnesium sulfate and concentrated to obtain a residue as a yellow oil.The residue was subjected to silica gel column chromatography with adeveloping solvent (ethyl acetate:hexane=1:1). As a result, the desired4-(7-carboxy-1,1-dimethyl-3-oxaheptyl)-5-(3-t-butyldimethylsiloxyphenyl)-3,3-dimethyl-2,3-dihydrofuran(compound (26)) was obtained as a colorless oil (475 mg, 0.997 mmol,72.3%).

¹H-NMR (500 MHz, CDCl₃): δ_(H) 0.18 (s, 6H), 0.98 (s, 9H), 1.04 (s, 6H),1.31 (s, 6H), 1.56–1.62 (m, 2H), 1.67–1.76 (m, 2H), 2.38 (t, J=7.0Hz,2H), 3.10 (s, 2H), 3.25 (t, J=6.5 Hz, 2H), 3.87 (s, 2H), 6.77–6.80 (m,2H), 6.90 (dd, J=8.0 and 1.5 Hz, 1H), 7.15–7.19 (m, 1H) ppm.

EXAMPLE 26

To a solution having5-(3-t-butyldimethylsiloxyphenyl)-4-(7-carboxy-1,1-dimethyl-3-oxaheptyl)-3,3-dimethyl-2,3-dihydrofuran(compound (26)) (394 mg, 0.827 mmol) dissolved in anhydrous acetonitrile(5 mL) in a nitrogen atmosphere at room temperature,di(N-succinimidyl)carbonate (318 mg, 1.241 mmol) and triethylamine (onedrop) were added, followed by stirring for 50 minutes. The reactionsolution was concentrated, and the concentrate was put into a silica gelcolumn and developed with a mixed solvent of hexane and ethyl acetate(1:1), whereby5-(3-t-butyldimethylsiloxyphenyl)-3,3-dimethyl-4-(1,1-dimethyl-7-succinimidoxycarbonyl-3-oxaheptyl)-2,3-dihydrofuran(compound (27)) was obtained as a colorless oil in an amount of 433 mgin a yield of 91.2%.

¹H-NMR (500 MHz, CDCl₃): δ_(H) 0.18 (s, 6H), 0.98 (s, 9H), 1.04 (s, 6H),1.31 (s, 6H), 1.61–1.67 (m, 2H), 1.81 (quintet, J=7.5 Hz, 2H), 2.64 (t,J=7.5 Hz, 2H), 2.84 (br-d, J=6.5 Hz, 4H), 3.10 (s, 2H), 3.26 (t, J=6.0Hz, 2H), 3.86 (s, 2H), 6.77–6.80 (m, 2H), 6.90 (dd, J=9.0 and 1.5 Hz,1H), 7.17 (t, J=8.0 Hz, 1H) ppm.

EXAMPLE 27

5-(3-t-butyldimethylsiloxyphenyl)-3,3-dimethyl-4-(1,1-dimethyl-7-succinimidoxycarbonyl-3-oxaheptyl)-2,3-dihydrofuran(compound (27)) (313 mg, 0.545 mmol) and TPP (2.5 mg) were added todichloromethane (15 mL), and the mixture was irradiated with visiblelight by a 940 W sodium lamp in an oxygen atmosphere at 0° C. for onehour. The reaction mixture was concentrated, and the concentrate was putinto a silica gel column and developed with a mixed solvent of hexaneand ethyl acetate (1:1), whereby1-(3-t-butyldimethylsiloxyphenyl)-4,4-dimethyl-5-(1,1-dimethyl-7-succinimidoxycarbonyl-3-oxaheptyl)-2,6,7-trioxabicyclo[3.2.0]heptane(compound (28)) was obtained in an amount of 276 mg in a yield of 83.6%.

1H-NMR (500 MHz, CDCl₃): δ_(H) 0.19 (s, 6H), 0.86 (s, 3H), 0.98 (s, 9H),1.14 (s, 3H), 1.17 (s, 3H), 1.38 (s, 3H), 1.60–1.66 (m, 2H), 1.78(quintet, J=8.0 Hz, 2H), 2.62 (t, J=7.0 Hz, 2H), 2.84 (br-d, J=6.0 Hz,4H), 3.24 (d, J=9.0 Hz, 1H), 3.30–3.35 (m, 3H), 3.81 (d, J=8.5 Hz, 1H),4.57 (d, J=8.5 Hz, 1H), 6.85–6.88 (m, 1H), 7.11 (s, 1H), 7.20–7.27 (m,2H) ppm.

EXAMPLE 28

To a solution having4-(13-carbonyl-1,1-dimethyl-3-oxatridecan-1-yl)-5-(3-hydroxyphenyl)-3,3-dimethyl-2,3-dihydrofuran(compound (21)) (553 mg, 1.24 mmol) dissolved in anhydrous DMF (6 mL) ina nitrogen atmosphere at room temperature, imidazole (257 mg, 3.77 mmol)and chlorinated t-butyldimethylsilane (566 mg, 3.76 mmol) were added,followed by stirring for 1.5 hours. The reaction mixture was put into asaturated ammonium chloride aqueous solution and extracted with ethylacetate. The organic layer was washed with a saturated sodium chlorideaqueous solution, dried over anhydrous magnesium sulfate and thenconcentrated. The concentrate was put into a silica gel column anddeveloped with a mixed solvent of hexane and ethyl acetate (2:1),whereby5-(3-t-butyldimethylsiloxyphenyl)-4-(13-carboxy-1,1-dimethyl-3-oxatridecan-1-yl)-3,3-dimethyl-2,3-dihydrofuran(compound (29)) was obtained as a colorless oil in an amount of 607 mgin a yield of 87.4%.

¹H-NMR (400 Hz, CDCl₃) δ_(H) 0.18 (s, 6H), 0.98 (s, 9H), 1.03 (s, 6H),1.27–1.30 (m, 18H), 1.47–1.52 (m, 2H), 1.63 (quintet, J=7.2 Hz, 2H),2.34 (t, J=7.6 Hz, 2H). 3.10 (s, 2H), 3.24 (t, J=6.8 Hz, 2H), 3.86 (s,2H), 6.76–6.79 (m, 2H), 6.90 (d with fine coupling, J=7.6 Hz, 1H), 7.16(t with fine coupling, J=7.2 Hz, 1H) ppm.

EXAMPLE 29

To a solution having5-(3-t-butyldimethylsiloxyphenyl)-4-(13-carboxy-1,1-dimethyl-3-oxatridecan-1-yl)-3,3-dimethyl-2,3-dihydrofuran(compound (29)) (405 mg, 0.722 mmol) dissolved in anhydrous acetonitrile(5 mL) in a nitrogen atmosphere at room temperature,di(N-succinimidyl)carbonate (237 mg, 0.925 mmol) and triethylamine (onedrop) were added, followed by stirring for 50 minutes. The reactionsolution was concentrated, and the concentrate was put into a silica gelcolumn and developed with a mixed solvent of hexane and ethyl acetate(2:1), whereby5-(3-t-butyldimethylsiloxyphenyl)-3,3-dimethyl-4-(1,1-dimethyl-13-succinimidoxycarbonyl-3-oxatridecan-1-yl)-2,3-dihydrofuran(compound (30)) was obtained as a colorless oil in an amount of 431 mgin a yield of 90.5%.

¹H-NMR (400 Hz, CDCl₃) δ_(H) 0.18 (s, 6H), 0.98 (s, 9H), 1.03 (s, 6H),1.28–1.31 (m, 18H), 1.49–1.54 (m, 2H), 1.74 (quintet, J=7.6 Hz, 2H),2.59 (t, J=7.6 Hz, 2H), 2.82 (s, 4H), 3.11 (s, 2H), 3.24 (t, J=6.8 Hz,2H), 3.86 (s, 2H), 6.76–6.79 (m, 2H), 6.90 (d, J=7.6 Hz, 1H), 7.16 (twith fine coupling, J=7.2 Hz, 1H) ppm.

EXAMPLE 30

5-(3-t-butyldimethylsiloxyphenyl)-3,3-dimethyl-4-(1,1-dimethyl-13-succinimidoxycarbonyl-3-oxatridecan-1-yl)-2,3-dihydrofuran(compound (30)) (177 mg, 0.269 mmol) and TPP (0.8 mg) were added todichloromethane (15 mL), and the mixture was irradiated with visiblelight by a 940 W sodium lamp in an oxygen atmosphere at 0° C. for 1.5hours. The reaction mixture was concentrated, and the concentrate wasput into a silica gel column and developed with a mixed solvent ofhexane and ethyl acetate (2:1), whereby1-(3-t-butyldimethylsiloxyphenyl)-4,4-dimethyl-5-(1,1-dimethyl-13-succinimidoxycarbonyl-3-oxatridecan-1-yl)-2,6,7-trioxabicyclo[3.2.0]heptane(compound (31)) was obtained in an amount of 181 mg in a yield of 97.5%.

¹H-NMR (400 Hz, CDCl₃) δ_(H) 0.19 (s, 6H), 0.87 (s, 3H), 0.98 (s, 9H),1.14 (s, 3H), 1.16 (s, 3H). 1.26–1.40 (m, 12H), 1.38 (s, 3H), 1.46–1.50(m, 2H), 1.74 (quintet, J=7.6 Hz, 2H), 2.60 (t, J=7.6 Hz, 2H), 2.82 (s,4H), 3.22–3.29 (m, 4H), 3.80 (d, J=8.4 Hz, 1H), 4.57 (d, J=8.0 Hz, 1H),6.84–6.87 (m, 1H), 7.11 (s, 1H), 7.12–7.25 (m, 2H) ppm.

EXAMPLE 31

To a solution having1-(3-t-butyldimethylsiloxyphenyl)-4,4-dimethyl-5-(1,1-dimethyl-13-succinimidoxycarbonyl-3-oxatridecan-1-yl)-2,6,7-trioxabicyclo[3.2.0]heptane(53.7 mg, 0.0778 mmol) (compound (31)) dissolved in anhydrousdichloromethane (1 mL) in a nitrogen atmosphere at 0° C.,β-phenetylamine (11 mg, 0.0908 mmol) dissolved in anhydrousdichloromethane (1 mL), was added, followed by stirring for one hour and20 minutes. To the reaction solution, β-phenetylamine (5 mg, 0.0413mmol) dissolved in anhydrous dichloromethane (0.5 mL), was furtheradded, followed by stirring for 30 minutes. The reaction mixture wasconcentrated, and the concentrate was put into a silica gel column anddeveloped with a mixed solvent of hexane and ethyl acetate (1:1),whereby1-(3-t-butyldimethylsiloxyphenyl)-4,4-dimethyl-5-[1,1-dimethyl-13-(2-phenylethylcarbamoyl)-3-oxatridecan-1-yl]-2,6,7-trioxabicyclo[3.2.0]heptane(compound (32)) was obtained in an amount of 51.8 mg in a yield of95.6%.

¹H-NMR (400 Hz, CDCl₃) δ_(H) 0.19 (s, 6H), 0.86 (s, 3H), 0.98 (s, 9H),1.14 (s, 3H), 1.16 (s, 3H), 1.24–1.40 (m, 12H), 1.38 (s, 3H), 1.45–1.50(m, 2H), 1.52–1.60 (m, 2H), 2.11 (t, J=7.6 Hz, 2H), 2.81 (t, J=6.8 Hz,2H), 3.22–3.29 (m, 4H), 3.52 (q, J=6.8 Hz, 2H), 3.80 (d, J=8.4 Hz, 1H),4.57 (d, J=8.0 Hz, 1H), 6.84–6.87 (m, 1H), 7.11 (s, 1H), 7.18–7.33 (m,7H) ppm.

EXAMPLE 32

To a solution having5-(3-acetoxyphenyl)-4-(13-carboxy-1,1-dimethyl-3-oxatridecan-1-yl)-3,3-dimethyl-2,3-dihydrofuran(192 mg, 0.393 mmol) (compound (22)) dissolved in anhydrous acetonitrile(3 mL) in a nitrogen atmosphere at room temperature,di(N-succinimidyl)carbonate (125 mg, 0.488 mmol) and triethylamine (onedrop) were added, followed by stirring for 1.5 hours. The reactionsolution was concentrated, and the concentrate was put into a silica gelcolumn and developed with a mixed solvent of hexane and ethyl acetate(2:1), whereby5-(3-acetoxyphenyl)-3,3-dimethyl-4-(1,1-dimethyl-13-succinimidoxycarbonyl-3-oxatridecan-1-yl)-2,3-dihydrofuran(compound (33)) was obtained as a colorless oil in an amount of 191 mgin a yield of 83.0%.

¹H-NMR (400 Hz, CDCl₃) δ_(H) 1.03 (s, 6H), 1.26–1.42 (m, 18H), 1.47–1.52(m, 2H), 1.73 (quintet, J=7.6 Hz, 2H), 2.27 (s, 3H), 2.59 (t, J=7.6 Hz,2H), 2.80 (s, 4H), 3.08 (s, 2H), 3.23 (t, J=6.8 Hz, 2H), 3.85 (s, 2H),7.03–7.09 (m, 2H), 7.19 (d with fine coupling, J=7.6 Hz, 1H), 7.31 (t,J=7.6 Hz, 1H) ppm.

EXAMPLE 33

5-(3-acetoxyphenyl)-3,3-dimethyl-4-(1,1-dimethyl-13-succinimidoxycarbonyl-3-oxatridecan-1-yl)-2,3-dihydrofuran(compound (33)) (100 mg, 0.171 mmol) and TPP (0.8 mg) were added todichloromethane (10 mL), and the mixture was irradiated with visiblelight by a 940 W sodium lamp in an oxygen atmosphere at 0° C. for onehour. The reaction mixture was concentrated, and the concentrate was putinto a silica gel column and developed with a mixed solvent of hexaneand ethyl acetate (3:2), whereby1-(3-acetoxyphenyl)-4,4-dimethyl-5-(1,1-dimethyl-13-succinimidoxycarbonyl-3-oxatridecan-1-yl)-2,6,7-trioxabicyclo[3.2.0]heptane(compound (34)) was obtained in an amount of 87 mg in a yield of 82.5%.

¹H-NMR (400 Hz, CDCl₃) δ_(H) 0.86 (s, 3H), 1.15 (s, 3H), 1.16 (s, 3H),1.24–1.40 (m, 12H), 1.39 (s, 3H), 1.46–1.50 (m, 2H), 1.74 (quintet,J=7.6 Hz, 2H), 2.30 (s, 3H), 2.60 (t, J=7.6 Hz, 2H), 2.83 (s, 4H), 3.23(s, 2H), 3.27 (t with fine coupling, J=6.8 Hz, 2H), 3.81 (d, J=8.0 Hz,1H), 4.58 (d, J=8.0 Hz, 1H), 7.12–7.15 (m, 1H), 7.37 (s, 1H), 7.40 (t,J=7.6 Hz, 1H), 7.51 (d, J=7.2 Hz, 1H) ppm.

EXAMPLE 34

To a solution having5-(3-t-butyl-4,4-dimethyl-4,5-dihydrofuran-2-yl)-2-(5-trifluoromethylisooxazol-3-yl)phenol(compound (35)) (500 mg, 1.31 mmol) as a known compound disclosed inJP-A-2002-338576, dissolved in pyridine (100 mL) at room temperature,DMAP (20 mg) and glutaric anhydride (1.496 g, 13.11 mmol) were added,followed by stirring for two hours at 100° C. The reaction mixture wasput into a 1N hydrochloric acid aqueous solution and extracted withethyl acetate. The organic layer was washed with a saturated sodiumchloride aqueous solution, dried over anhydrous magnesium sulfate andthen concentrated. The concentrate was put into a silica gel column anddeveloped with a mixed solvent of hexane and ethyl acetate (1:1),whereby5-(3-t-butyl-4,4-dimethyl-4,5-dihydrofuran-2-yl)-2-(5-trifluoromethylisooxazol-3-yl)phenylgultarate (compound (36)) was obtained as a white solid in an amount of510 mg in a yield of 87.5%.

¹H-NMR (500 Hz, CDCl₃) δ_(H) 1.07 (s, 9H), 1.34 (s, 6H), 2.11 (quintet,J=7.0 Hz, 2H), 2.57 (t, J=7.5 Hz, 2H), 2.79 (t, J=7.5 Hz, 2H), 3.89 (s,2H), 7.16 (d, J=1.0 Hz, 1H), 7.34 (dd, J=8.3 and 1.5 Hz, 1H), 7.55 (d,J=1.0 Hz, 1H), 8.10 (d, J=7.5 Hz, 1H) ppm.

EXAMPLE 35

To a solution having5-(3-t-butyl-4,4-dimethyl-4,5-dihydrofuran-2-yl)-2-(5-trifluoromethylisooxazol-3-yl)phenylgultarate (compound (36)) (510 mg, 1.03 mmol) dissolved in DMF (10 mL)at 0° C., N-hydroxysuccinimide (179 mg, 1.55 mmol) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (302 mg,1.57 mmol) were added, followed by stirring at 4° C. overnight. Thereaction mixture was put into a saturated sodium chloride aqueoussolution and extracted with ethyl acetate. The organic layer was washedwith a saturated sodium chloride aqueous solution, dried over anhydrousmagnesium sulfate and then concentrated. The concentrate was put into asilica gel column and developed with a mixed solvent of hexane and ethylacetate (1:1), whereby5-(3-t-butyl-4,4-dimethyl-4,5-dihydrofuran-2-yl)-2-(5-trifluoromethylisooxazol-3-yl)phenylsuccinimidylgultarate (compound (37)) was obtained as a white solid in an amount of515 mg in a yield of 84.4%.

¹H-NMR (500 Hz, CDCl₃) δ_(H) 1.07 (s, 9H), 1.34 (s, 6H), 2.21 (quintet,J=7.0 Hz, 2H), 2.83–2.87 (m, 8H), 3.89 (s, 2H), 7.17 (d, J=1.0 Hz, 1H),7.34 (dd, J=8.3 and 1.5 Hz, 1H), 7.63 (d, J=1.0 Hz, 1H), 8.10 (d, J=8.5Hz, 1H) ppm.

EXAMPLE 36

5-(3-t-butyl-4,4-dimethyl-4,5-dihydrofuran-2-yl)-2-(5-trifluoromethylisooxazol-3-yl)phenylsuccinimidylgultarate (compound (37)) (505 mg, 0.85 mmol) and TPP (15 mg) were addedto dichloromethane (15 mL), and the mixture was irradiated with visiblelight by a 940 W sodium lamp in an oxygen atmosphere at 0° C. for twohours. The reaction mixture was concentrated, and the concentrate wasput into a silica gel column and developed with a mixed solvent ofhexane and ethyl acetate (1:1), whereby5-(5-t-butyl-4,4-dimethyl-2,6,7-trioxabicyclo[3.2.0]hept-1-yl)-2-(5-trifluoromethylisooxazol-3-yl)phenylsuccinimidylgultarate (compound (38)) was obtained as a white solid in an amount of445 mg in a yield of 83.6%.

¹H-NMR (500 Hz, CDCl₃) δ_(H) 1.00 (s, 9H), 1.17 (s, 3H), 1.39 (s, 3H),2.22 (quintet, J=7.0 Hz, 2H), 2.83–2.89 (m, 8H), 3.87 (d, J=8.0 Hz, 1H),4.60 (d, J=8.0 Hz, 1H), 7.49 (s, 1H), 7.65 (dd, J=1.5 Hz, 1H), 7.68 (d,J=9.5 Hz, 1H), 8.17 (d, J=8.0 Hz, 1H) ppm.

EXAMPLE 37

A thyroid stimulating hormone antibody (TSH antibody) was put into adialysis tube, and dialysis was carried out at from 2 to 8° C. against a0.1 M phosphate buffer solution (pH 7.0). Using 1 L of the buffersolution per operation, dialysis was carried out for at least 4 hours.After completion of the dialysis, the antibody solution was put into asyringe provided with a 0.45 μm filter, and filtration was carried out.The volume at that time was 0.45 mL, and the concentration was 17.7mg/mL. Then, a 0.1 M phosphate buffer solution (pH 7.0) was added sothat the protein concentration in this antibody solution became 5 mg/mL.This solution was transferred to a reactor and immersed in a constanttemperature vessel controlled at a temperature of 4±1° C. for 30minutes. 0.015 mL of a DMF solution (concentration: 10.4 mg/mL) of5-(5-t-butyl-4,4-dimethyl-2,6,7-trioxabicyclo[3.2.0]hept-1-yl)-2-(5-trifluoromethylisooxazol-3-yl)phenylsuccinimidylgultarate (compound (38)) was added. After completion of the dropwiseaddition, the mixture was gently stirred by means of a vortex, and thenstirring was stopped, and the mixture was left to stand at 4±1° C. for17 hours in the constant temperature vessel. After completion of thereaction, the reaction solution was transferred to a dialysis tube, anddialysis was carried out at from 2 to 8° C. against a 0.1 M phosphoricacid/NaCl buffer solution (0.1% NaN₃) (pH 7.0). Using at least 1 L ofthe buffer solution per operation, dialysis was carried out three timesfor at least 4 hours. After completion of the dialysis, the antibodysolution was put into a syringe provided with a 0.22 μm filter, andfiltration was carried out. As a result, 0.88 mL of a chemiluminescentsubstrate-labeled thyroid stimulating hormone antibody was obtained, andits concentration was 7,599 mA.

EXAMPLE 38

The chemiluminescent substrate-labeled thyroid stimulating hormoneantibody prepared in Example 37, was diluted with a 20 mM ACES (0.1%BSA, 0.1% NaN₃) buffer solution (pH 6.5) so that the concentration wouldbe 100 mA. To 33 μL of this solution, 200 μL of a 0.5 N sodium hydroxideaqueous solution was added as a trigger, and the luminescence wasmeasured by means of a luminescence measuring apparatus. The obtainedluminescence curve is shown in FIG. 1.

The 1,2-dioxetane derivatives (I) and (III) of the present invention arestable as compounds themselves, whereby handling is easy.

Further, the 1,2-dioxetane derivatives (I) and (III) of the presentinvention can be labeled, for example, to an organic compound or abiological molecule via a part of the group such as X or W in theirstructures. Accordingly, by bonding them to a substance having aspecific affinity, it is possible to obtain an immunoassay reagent ofthe present invention. By using the immunoassay reagent of the presentinvention, it is possible to lower the background at the time of themeasurement by e.g. an immunoassay. By this effect, measurement withhigher sensitivity in e.g. an immunoassay, will be possible.

Further, in the case of a 1,2-dioxetane derivative (I) wherein Z is—OSi(R₇R₈)— (wherein each of R₇ and R₈ which are independent of eachother, is an alkyl group or an aryl group) or —(R₉R₁₀)SiO— (wherein eachof R₉ and R₁₀ which are independent of each other, is an alkyl group oran aryl group), and in the case of a 1,2-dioxetane derivative (III), ifimmunoassay reagents of the present invention are obtained by usingthem, and they are used to form and detect an immunoreaction product ona solid phase, there will be the following effects. Namely, the portioncorresponding to the 1,2-dioxetane derivative in the immunoreactionproduct formed on the solid phase will be decomposed in the presence offluorine ions or under an alkaline condition, whereby by virtue of thecharacteristic due to its structure, the portion having the1,2-dioxetane structure will be decomposed in such a manner that will becleaved from the immunoreaction product on the solid phase and it willbe discharged into the solution, whereupon luminescence will take placeat the same time. Accordingly, the luminescence will take place in thesolution i.e. not on the solid phase. Thus, the detection of theluminescence is easy with no substantial noise, whereby measurement withhigh sensitivity will be possible. Further, the luminescence will not bedetected on the solid phase, whereby detection will not be fluctuated bythe shape of the solid phase. Thus, the system will be suitable fordetecting the luminescence in the liquid phase (homogeneous system)without loss.

The entire disclosures of Japanese Patent Application No. 2002-64040filed on Mar. 8, 2002, Japanese Patent Application No. 2002-88380 filedon Mar. 27, 2002 and Japanese Patent Application No. 2003-16454 filed onJan. 24, 2003 including specifications, claims, drawings and summariesare incorporated herein by reference in their entireties.

1. A chemiluminescent reagent, comprising: a 1,2-dioxetane derivative ofthe formula (I):

wherein Ar is an aryl group which may have an alkyl group, an arylgroup, a halogen atom, an alkoxyl group, a carboxyl group, a formylgroup, an alkyl ester, an aryl ester, an alkylketone, an arylketone or ahetero ring bonded thereto, X is a substituent capable of labeling anorganic compound or a biological molecule, or an ester, Y is a hydrogenatom, an acyl group or a group of the formula —Si(R₄R₅R₆), wherein eachof R₄, R₅ and R₆ which are independent of one another, is an alkyl groupor an aryl group, Z is an alkyl group, an aryl group, an oxygen atom, asulfur atom, a carbonyl group, —(CO)—O—, —O—(CO)—, —NH—, —NH—CO—,—CO—NH—, —OSi(R₇R₈)—, wherein each of R₇ and R₈ which are independent ofeach other, is an alkyl group or aryl group; or a group of the formula—(R₉R₁₀)SiO—, wherein each of R₉ and R₁₀ which are independent of eachother, is an alkyl group or an aryl group; each of R₁ and R₂ is an alkylgroup or an aryl group, and R₃ is a spacer.
 2. An immunoassay reagent,comprising a 1,2-dioxetane derivative of formula (I) bonded via X offormula (I) to an antibody or a receptor:

wherein Ar is an aryl group which may have an alkyl group, an arylgroup, a halogen atom, an alkoxyl group, a carboxyl group, a formylgroup, an alkyl ester, an aryl ester, an alkylketone, an arylketone or ahetero ring bonded thereto, X is a substituent capable of labeling anorganic compound or a biological molecule, or an ester, Y is a hydrogenatom, an acyl group or a group of the formula —Si(R₄R₅R₆), wherein eachof R₄, R₅ and R₆ which are independent of one another, is an alkyl groupor an aryl group, Z is an alkyl group, an aryl group, an oxygen atom, asulfur atom, a carbonyl group, —(CO)—O—, —O—(CO)—, —NH—, —NH—CO—,—CO—NH—, —OSi(R₇R₈)—, wherein each of R₇ and R₈ which are independent ofeach other, is an alkyl group or aryl group; or a group of the formula—(R₉R₁₀)SiO—, wherein each of R₉ and R₁₀ which are independent of eachother, is an alkyl group or an aryl group; each of R₁ and R₂ is an alkylgroup or an aryl group, and R₃ is a spacer.
 3. An immunoassay reagent,comprising: a 1,2-dioxetane derivative of formula (II) bonded via W offormula (II) to an antibody or a receptor:

wherein Y is a hydrogen atom, an acyl group or a group of the formula—Si(R₄R₅R₆), wherein each of R₄, R₅ and R₆ which are independent of oneanother, is an alkyl group or an aryl group, n is an integer of from 1to 20, W is a hydrogen atom, an alkyl group or a succinimidosubstituent, and U is a hydrogen atom, an alkyl group, an aryl group, ahalogen atom, an alkoxyl group, a carboxyl group, a formyl group, analkyl ester, an aryl ester, an alkylketone, an arylketone or a heteroring.
 4. A method of detecting a substance, comprising: mixing theimmunoassay reagent according to claim 2 and said substance to bedetected or a mixture containing said substance to be detected, toobtain a reaction mixture; reacting said reaction mixture to bond saidsubstance to be detected with said immunoassay reagent; and measuringthe amount of immunoassay reagent which was bonded or not bonded to saidsubstance to be detected, wherein said immunoassay reagent is decomposedin the presence of fluorine ions or under an alkaline condition, whereinsaid decomposition is accompanied by chemiluminescence, and an intensityof said chemiluminescence is measured.
 5. The method according to claim4, wherein said substance to be detected is selected from the groupconsisting of hormones, cancer-related substances, viral antigens, viralantibodies and nucleic acids.
 6. The method according to claim 4,wherein said substance to be detected is selected from the groupconsisting of hCG, TSH, LH, AFP, CEA, HIV, HTLV-I, DNA and RNA.
 7. Amethod of detecting a substance, comprising: mixing the immunoassayreagent according to claim 3 and said substance to be detected or amixture containing said substance to be detected, to obtain a reactionmixture; reacting said reaction mixture to bond said substance to bedetected with said immunoassay reagent; and measuring the amount ofimmunoassay reagent which was bonded or not bonded to said substance tobe detected, wherein said immunoassay reagent is decomposed in thepresence of fluorine ions or under an alkaline condition, wherein saiddecomposition is accompanied by chemiluminescence, and an intensity ofsaid chemiluminescence is measured.
 8. The method according to claim 7,wherein said substance to be detected is selected from the groupconsisting of hormones, cancer-related substances, viral antigens, viralantibodies and nucleic acids.
 9. The method according to claim 7,wherein said substance to be detected is selected from the groupconsisting of hCG, TSH, LH, AFP, CEA, HIV, HTLV-I, DNA and RNA.